Modified gram positive bacteria and uses thereof

ABSTRACT

The present invention relates to a gram positive bacterium, preferably a lactic acid bacterium (LAB) or  Bifidobacterium , with increased stress resistance and/or improved manufacturing, processing and/or storage characteristics. In particular, the invention relates to a gram positive bacterium which accumulate intracellular trehalose. The gram positive bacterium according to the invention lack trehalose 6-phosphate phosphorylase (TrePP) activity. The gram positive bacterium may further lack cellobiose-specific PTS system IIC component (ptcC) activity. The gram positive bacterium may further overexpress trehalose transporters. The invention further relates to compositions comprising such gram positive bacterium as well as methods and uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. patent applicationSer. No. 14/346,504, filed Mar. 21, 2014, the entirety of which isincorporated herein by reference. U.S. patent application Ser. No.14/346,504 is the U.S. National Phase under 35 U.S.C. § 371 ofInternational Application PCT/EP2012/068633, filed Sep. 21, 2012, whichclaims priority to EP 11182642.6, filed Sep. 23, 2011.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 6, 2016, isnamed 205350_0024_ST25.txt and is 34,253 bytes in size.

FIELD OF THE INVENTION

The present invention relates to microorganisms, such as gram positivebacteria, with improved stress resistance and improved manufacturing,processing and storage characteristics. The invention in particularrelates to genetically modified microorganisms which accumulateintracellular trehalose. The invention further relates to uses of thesemicroorganisms in food technology and medical applications.

BACKGROUND OF THE INVENTION

Gram-positive bacteria are collectively classified as having a singlelipid bilayer plasma membrane. Gram positive bacteria include amultitude of bacilliform and cocciform bacterial genera, among whichBifidobacteria and a group of genera collectively known as lactic acidbacteria (LAB). LAB comprise a clade of Gram-positive, low-GC,acid-tolerant, generally non-sporulating, non-respiring rod or coccithat are associated by their common metabolic and physiologicalcharacteristics. These bacteria, usually found in (decomposing) plantsand dairy products, produce lactic acid as the major metabolicend-product of carbohydrate fermentation. This trait has, throughouthistory, linked LAB with food fermentations, as acidification inhibitsthe growth of spoilage agents. A prototype LAB Lactococcus lactis is amesophilic and microaerophilic fermenting lactic acid bacterium. Whilethe bacterium is extensively used in food fermentations, especially inthe dairy industry, there is an increasing interest for its use inmedicaments and nutraceuticals, as medication to treat infections inbodily cavities such as vaginal infections, or as carrier for thedelivery of biological active molecules. In all those cases, there is aneed for highly viable starter cultures, or pharmaceutical ornutraceutical formulations comprising a high proportion of viablebacteria. L. lactis, however, tends to lose viability during storage, orduring processing (for a.o the production of a dry powder formula,tablet formation, . . . ). The drop in viability is even more pronouncedwhen the bacterium after lyophilisation is submitted to additionalstress such as high acidity or the presence of bile salts.

Several methods have been proposed to overcome this problem. The use oftrehalose is of particular interest. Trehalose(α-D-glucopyranosyl-1,1-α-D-glucopyranoside) is a non-reducingdisaccharide that occurs in a large variety of organisms, ranging frombacteria to invertebrate animals. Trehalose, sometimes in combinationwith dextran, is often used as and externally added cryopreservant.Externally added trehalose functions as a saccharide matrix (Conrad etal., 2000), and exerts it protective effect especially during freezedrying, where it acts as a glass former. Moreover, trehalose is wellrecognized as stress metabolite, and it has been extensively studied infungi, especially in Saccharomyces cerevisiae. High concentrations ofinternal trehalose do improve the storage capacity and result in ahigher viability upon cryopreservation. However, it is important to notethat externally added trehalose rarely leads to internal trehaloseaccumulation in micro-organisms, either because it is not taken up, orit is metabolized rapidly after uptake.

Termont et al. (Appl Environ Microbiol 72:7694; 2006) reported thatde-novo synthesized trehalose, through plasmid driven overexpression ofotsA (trehalose-6-phosphate synthase) and otsB (trehalose-6-phosphatephosphatase) accumulates intracellularly in L. lactis. Intracellulartrehalose accumulation but not exogenously added trehalose protects L.lactis from bile lysis and cell death through freeze-drying. As L.lactis is extremely sensitive, protection to bile lysis can be used as asuperb functional assay of intracellular trehalose accumulation.

Andersson et al. (J Biol Chem 276:42707; 2001) have described a novelpathway for trehalose utilization in L. lactis. This pathway employs theactivity of trehalose-6-phosphate phosphorylase (trePP), convertingtrehalose-6-phosphate to β-glucose 1-phosphate and glucose 6-phosphate.They describe insertional inactivation of trePP in L. lactis, resultingin loss of capacity to grow on trehalose.

For the intracellular accumulation of trehalose, Carvalho et al. (ApplEnviron Microbiol 77:4189; 2011) describe a method that makes use ofplasmid driven overexpression of L. lactis trePP andβ-phosphoglucomutase (pgmB). As indicated by these authors, given thatthe bacteria lack trehalose 6-phosphate phosphatase, the respectivegene, otsB, from food-grade organism P. freudenreichii was used toprovide the required activity. The resulting cells showed improvedresistance to cold shock, heat shock and acidity.

Although these processes certainly lead to an improvement of thestorage, there is a further need of methods that can lead to an improvedstorage of gram positive bacteria, such as LAB or Bifidobacteria, notonly in those cases where the bacterium is used for the delivery ofbiological active compounds in medical applications, but also when thebacterium is used in the food industry, such as the dairy industry.

SUMMARY OF THE INVENTION

Intracellular trehalose can protect microorganisms such as lactic acidbacteria (LAB), for instance Lactococcus lactis cells, from variousdetrimental agents or conditions. Examples are bile acid lysis,experienced by live LAB during intestinal transit, or freezing and/ordrying stress during freezing, drying, spray drying, lyophilization, asused for preservation of LAB.

Only a limited number of approaches are available that allow for theaccumulation of trehalose inside the cell. These make use of plasmiddriven overexpression of homologous or heterologous genes. This ishowever not a desirable configuration for use in pharmaceutical or foodproducts.

Here we report a novel approach that allows for the intracellularaccumulation of trehalose, based merely on the absence of trehalose6-phosphate phosphorylase (TrePP) activity in gram positive bacteria,preferably through rendering the gene encoding endogenous TrePPpartially or completely deleted, disrupted or inactivated such as beingincapable of producing functional trePP gene product. In contrast to thepresent approach, previous works (WO 2006/018446) taught to expressheterologous trehalose 6-phosphate phosphatase such as otsB to achievetrehalose accumulation. Carvalho et al. 2011 (supra) even instructed tooverexpress TrePP to obtain intracellular trehalose accumulation.Moreover, although LAB such as Lactococcus lactis may be able to utilizetrehalose, up to now no trehalose synthesizing Lactococcus lactis strainhas been described. No endogenous trehalose-6-phosphate synthase andtrehalose-6-phosphate phosphatase genes have been identified, which werebelieved to be a prerequisite for trehalose production starting fromglucose-6-phosphate, a metabolite present in L. lactis.

In an aspect, the invention relates to a gram positive bacterium,preferably a lactic acid bacterium (LAB) or Bifidobacterium, lackingtrehalose 6-phosphate phosphorylase (TrePP) activity for use as amedicament.

A further aspect provides a medicament comprising a gram positivebacterium, preferably a lactic acid bacterium (LAB) or Bifidobacterium,lacking TrePP activity. Such medicaments may for example encompasspharmaceutical formulations, nutraceuticals, medical foods or functionalfoods, or probiotics.

In another aspect, the invention provides the use of a gram positivebacterium, preferably a lactic acid bacterium (LAB) or Bifidobacterium,lacking TrePP activity as a probiotic or food additive, morespecifically as a non-medicinal probiotic or food additive. Withoutlimitation, such food additive may be a starter culture, preferably astarter culture for the preparation of a food product. Hence, a relatedaspect provides the use of a gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, lacking TrePP activityas a starter culture, preferably a starter culture for the preparationof a food product, more particularly wherein the food product is anon-medicinal food product.

A further aspect provides a probiotic composition or food additive, morespecifically a non-medicinal probiotic composition or food additive,comprising a gram positive bacterium, preferably a lactic acid bacterium(LAB) or Bifidobacterium, lacking TrePP activity. Without limitation,such food additive may be a starter culture, preferably a starterculture for the preparation of a food product. Hence, a related aspectprovides a starter culture, preferably a starter culture for thepreparation of a food product, comprising a gram positive bacterium,preferably a lactic acid bacterium (LAB) or Bifidobacterium, lackingTrePP activity.

Also provided by an aspect of the invention is a method for preparing afood product, comprising admixing a gram positive bacterium, preferablya lactic acid bacterium (LAB) or Bifidobacterium, lacking TrePPactivity, or said food additive or said starter culture with a substratematerial that is capable of being fermented by the gram positivebacterium, preferably a lactic acid bacterium (LAB) or Bifidobacterium.In embodiments, such method may further comprise the step of fermentingsaid substrate material. As well provided is thus a food productobtainable by any such method. A food product may encompass withoutlimitation probiotics.

Another aspect provides a method for preparing a medicament, such as apharmaceutical formulation, nutraceutical, medical food or functionalfood or probiotic, or for preparing a probiotic composition or foodadditive, more specifically a non-medicinal probiotic composition orfood additive, or for preparing a starter culture, preferably a starterculture for the preparation of a food product, comprising the steps of:i) propagating a gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, lacking TrePP activity in a mediumcomprising a substrate material capable of being fermented by said grampositive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, and ii) formulating the so propagated gram positivebacterium, preferably a lactic acid bacterium (LAB) or Bifidobacterium,into, respectively, the medicament or probiotic composition or foodadditive or starter culture. Hence, also covered is the use of a grampositive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, lacking TrePP activity for the preparation of amedicament, such as a pharmaceutical formulation, nutraceutical, medicalfood or functional food or probiotic, or for preparation of a probioticcomposition or food additive, more specifically a non-medicinalprobiotic composition or food additive, or for preparation of a starterculture, preferably a starter culture for the preparation of a foodproduct.

The inventors have found that gram positive bacteria, such as LAB orBifidobacteria, as described herein not only are capable ofintracellular trehalose accumulation, even independent of the carbonsource, but also that the gram positive bacterium show greatly enhancedresistance to various stress- and storage-associated conditions. Forexample, the gram positive bacterium are more resistant tostorage-associated manipulations, such as drying, freezing, spraydrying, or freeze-drying (lyophilisation). The gram positive bacteriumalso display enhanced survival, independent of the feeding or fastingstatus, in the gastro-intestinal system, indicating improved resistanceto acidity and bile lysis. The performance of the gram positivebacterium as described herein, whether in a medicinal setting or in thefood industry, is more reproducible than previously known. Hence thegram positive bacterium embodying the principles of the inventionprovide for a more robust environmental as well as bio-resistance.

In an aspect, the invention thus also relates to a method for internallyaccumulating trehalose in a gram positive bacterium, preferably a lacticacid bacterium (LAB) or Bifidobacterium, comprising propagating a grampositive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, lacking TrePP activity in a medium comprising asubstrate material capable of being fermented by said gram positivebacterium.

In a further aspect, the invention relates to a method for improvingstress resistance or manufacturing, processing and/or storagecharacteristics of a gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, comprising modifying the grampositive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, such as to lack TrePP activity. Preferably, the stressresistance or manufacturing, processing and/or storage characteristicsmay be one or more selected from the group comprising resistance to acidconditions, resistance to bile salts, resistance to heat, resistance tosalt, resistance to drying, freezing, spray-drying, or freeze-drying,and osmotic resistance.

Preferably, in the aforementioned gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, lacking TrePP activity,the gene encoding endogenous TrePP has been partially or completelydeleted, disrupted or inactivated such as being incapable of producingfunctional trePP gene product. It shall be appreciated that suchdeletion, disruption or inactivation may target for example the codingsequence of the trePP gene and/or the promoter from which trePP isexpressed.

In preferred embodiments, the gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, lacking TrePP activityas employed herein does not contain functional heterologous trehalose6-phosphate phosphatase. As already explained, overexpression ofheterologous trehalose 6-phosphate phosphatase, such as for example otsBfrom Escherichia coli or from Propionibacterium freudenreichii, waspreviously presumed to be requisite for achieving trehalose accumulationin gram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium.

In preferred embodiments, the gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, lacking TrePP activityas employed herein does not contain functional heterologous trehalose6-phosphate synthase. As already explained, overexpression ofheterologous trehalose 6-phosphate synthase, such as for example otsAfrom Escherichia coli, was previously indicated for achieving trehaloseaccumulation in LAB.

In preferred embodiments, the gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, lacking TrePP activityas employed herein does not contain functional heterologous trehalose6-phosphate phosphatase and does not contain functional heterologoustrehalose 6-phosphate synthase.

In preferred embodiments, the gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, lacking TrePP activityas employed herein may lack cellobiose-specific PTS system II Ccomponent (ptcC) activity. Preferably, in such gram positive bacterium,preferably a lactic acid bacterium (LAB) or Bifidobacterium, alsolacking ptcC activity, the gene encoding endogenous ptcC has beenpartially or completely deleted, disrupted or inactivated such as beingincapable of producing functional ptcC gene product. It shall beappreciated that such deletion, disruption or inactivation may targetfor example the coding sequence of the ptcC gene and/or the promoterfrom which ptcC is expressed. The inventors have unexpectedly observedthat over time the accumulated trehalose leaks to some extent out of thecells through an up to now unanticipated and unidentified trehalose exitport, whereby the trehalose can be detected in the supernatant.Surprisingly, they found that inactivation of ptcC prevents release oftrehalose. These findings are all the more unexpected, because ptcC upto now has never been associated with trehalose transport, and has notbeen suggested as a trehalose exit port responsible for trehaloseleakage and release into the surroundings. Also surprisingly, the knownand characterized trehalose transporters do not seem responsible forthis mechanism of trehalose leakage.

In preferred embodiments, the gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, lacking TrePP activityas employed herein may overexpress one or more trehalose transporters,preferably endogenous trehalose transporters, such as one or morephosphotransferase system genes comprised in the trehalose operon. Theinventors have surprisingly found that such overexpression, in contrastto the native trehalose induced expression, further enhances thecapacity of the gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, to accumulate and/or retainintracellular trehalose.

To recap, in some embodiments the gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, lacking TrePP activityas employed herein may additionally display any one, any two or allthree of the following characteristics: (a) the gram positive bacteriumdoes not contain functional heterologous trehalose 6-phosphatephosphatase; (b) the gram positive bacterium lacks ptcC activity; (c)the gram positive bacterium overexpresses one or more trehalosetransporters. In preferred embodiments, the gram positive bacteriumlacking TrePP activity may additionally display characteristic (a), ormore preferably may additionally display characteristics (a) and (b), ormay even more preferably additionally display characteristics (b) and(c), or may very preferably additionally display characteristics (a) and(b) and (c).

In this connection, a further aspect of the present invention is a grampositive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, lacking TrePP activity and further displaying any oneor preferably both of the following characteristics: (i) the grampositive bacterium lacks ptcC activity; (ii) the gram positive bacteriumoverexpresses one or more trehalose transporters.

A related aspect concerns a LAB lacking TrePP activity and notcontaining functional heterologous trehalose 6-phosphate phosphatase,and further displaying any one or preferably both of the followingcharacteristics: (i) the LAB lacks ptcC activity; (ii) the LABoverexpresses one or more trehalose transporters.

In preferred embodiments, the gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, as disclosed or employedherein may additionally contain one or more heterologous gene product.In some preferred embodiments, particularly wherein the gram positivebacterium, preferably a lactic acid bacterium (LAB) or Bifidobacterium,is intended for a medicinal use, such gene product(s) may beprophylactic and/or therapeutic gene product(s) or antigen(s).

In this connection, a further aspect of the present invention is a grampositive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, lacking TrePP activity and additionally containing oneor more heterologous gene products, preferably prophylactic and/ortherapeutic gene product(s). This gram positive bacterium may furtheroptionally display any one or preferably both of the followingcharacteristics: (i) the gram positive bacterium lacks ptcC activity;(ii) the gram positive bacterium overexpresses one or more trehalosetransporters.

A related aspect is a gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, lacking TrePP activity and notcontaining functional heterologous trehalose 6-phosphate phosphatase andadditionally containing one or more heterologous gene products,preferably prophylactic and/or therapeutic gene product(s). This grampositive bacterium may further optionally display any one or preferablyboth of the following characteristics: (i) the gram positive bacteriumlacks ptcC activity; (ii) the gram positive bacterium overexpresses oneor more trehalose transporters.

In certain embodiments, the gram positive bacterium, preferably a lacticacid bacterium (LAB) or Bifidobacterium, or medicament or food additiveor probiotic composition or starter culture as disclosed or employedherein may be dried, frozen, spray-dried, or freeze-dried (lyophilized).Accordingly, in some embodiments, any of the aforementioned methods forpreparing a medicament, or for preparing a food additive, or forpreparing a starter culture, or for preparing a probiotic compositions,or for internally accumulating trehalose in a gram positive bacterium,preferably a lactic acid bacterium (LAB) or Bifidobacterium, or forimproving stress resistance or manufacturing, processing and/or storagecharacteristics of a gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, may further comprise drying,spray-drying, freezing or freeze-drying (lyophilizing) the LAB,medicament, food additive, probiotic composition, or starter culture.

In certain embodiments of the aforementioned method for preparing amedicament, or for preparing a food additive, or for preparing a starterculture, or in certain embodiments of the aforementioned method forinternally accumulating trehalose in the gram positive bacterium,preferably a lactic acid bacterium (LAB) or Bifidobacterium, the culturemedium may comprise maltose or glucose or a combination of maltose andglucose, as a carbon source, preferably as main or even sole carbonsource. In certain embodiments, the culture medium substantially doesnot contain externally (exogenously) added trehalose. The inventors havesurprisingly found that gram positive bacteria, such as lactic acidbacteria (LAB) or Bifidobacteria, as disclosed herein have acquired thecapacity to utilize carbon sources such as maltose or glucose toaccumulate trehalose inside the cells. Accordingly, the gram positivebacteria according to the invention can advantageously be grown on forinstance maltose as the sole carbon source, which is cheaper thantrehalose, yet will accumulate intracellular trehalose. Nevertheless, itshall be appreciated that in certain embodiments, the culture medium maycontain externally (exogenously) added trehalose.

The above and further aspects and preferred embodiments of the inventionare described in the following sections and in the appended claims. Thesubject matter of appended claims is hereby specifically incorporated inthis specification.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Intracellular trehalose accumulation is possible following trePPinactivation, following otsB expression or a combination thereof.

FIG. 2: The accumulation of exogenous trehalose in L. lactis cellsprovides protection towards bile lysis. (A) survival; and (B) trehalosecontent.

FIG. 3: Accumulation and stability of intracellular trehalose. (A)trehalose release over time; and (B) trehalose increase in supernatant.

FIG. 4: Trehalose accumulation and release in various strains describedin Table 2. Strains were supplemented with 100 mM (A) or 500 mM (B)trehalose.

FIG. 5: Inactivation of ptcC prevents (in M9 salts, panel A) or delays(in 0.5% oxgal, panel B) the release of intracellular trehalose.

FIG. 6: The accumulation of exogenous trehalose in L. lactis cellsprovides protection towards bile lysis. (A) release of intracellulartrehalose over time; and (B) survival over time in 0.5% oxgal

FIG. 7: trePP KO strains (both ptcC wt as well as ptcC KO) are capableof converting glucose or maltose to intracellular trehalose

FIG. 8: Enhanced survival during intestinal passage through porcineintestine, both when pigs were fasted for 24 hours (A) as well as duringad libitum food availability (B).

FIG. 9: Trehalose accumulation after production of biomass.

FIG. 10: Stimulation of the accumulation of intracellular trehalose bymaltose.

FIG. 11: Conversion of maltose to intracellular trehalose during orafter production of biomass.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms “a”, “an”, and “the” include bothsingular and plural referents unless the context clearly dictatesotherwise.

The terms “comprising”, “comprises” and “comprised of” as used hereinare synonymous with “including”, “includes” or “containing”, “contains”,and are inclusive or open-ended and do not exclude additional,non-recited members, elements or method steps. It will be appreciatedthat the terms “comprising”, “comprises” and “comprised of” as usedherein comprise the terms “consisting of”, “consists” and “consists of”,as well as the terms “consisting essentially of”, “consists essentially”and “consists essentially of”.

The recitation of numerical ranges by endpoints includes all numbers andfractions subsumed within the respective ranges, as well as the recitedendpoints.

The term “about” or “approximately” as used herein when referring to ameasurable value such as a parameter, an amount, a temporal duration,and the like, is meant to encompass variations of +/−20% or less,preferably +/−10% or less, more preferably +/−5% or less, and still morepreferably +/−1% or less of and from the specified value, insofar suchvariations are appropriate to perform in the disclosed invention. It isto be understood that the value to which the modifier “about” or“approximately” refers is itself also specifically, and preferably,disclosed.

Whereas the terms “one or more” or “at least one”, such as one or moreor at least one member(s) of a group of members, is clear per se, bymeans of further exemplification, the term encompasses inter alia areference to any one of said members, or to any two or more of saidmembers, such as, e.g., any or >7 etc. of said members, and up to allsaid members.

All references cited in the present specification are herebyincorporated by reference in their entirety. In particular, theteachings of all references herein specifically referred to areincorporated by reference.

Unless otherwise defined, all terms used in disclosing the invention,including technical and scientific terms, have the meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. By means of further guidance, term definitions are included tobetter appreciate the teaching of the present invention.

In the following passages, different aspects of the invention aredefined in more detail. Each aspect so defined may be combined with anyother aspect or aspects unless clearly indicated to the contrary. Inparticular, any feature indicated as being preferred or advantageous maybe combined with any other feature or features indicated as beingpreferred or advantageous.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, appearances of the phrases“in one embodiment” or “in an embodiment” in various places throughoutthis specification are not necessarily all referring to the sameembodiment, but may. Furthermore, the particular features, structures orcharacteristics may be combined in any suitable manner, as would beapparent to a person skilled in the art from this disclosure, in one ormore embodiments. Furthermore, while some embodiments described hereininclude some but not other features included in other embodiments,combinations of features of different embodiments are meant to be withinthe scope of the invention, and form different embodiments, as would beunderstood by those in the art. For example, in the appended claims, anyof the claimed embodiments can be used in any combination.

In the following detailed description of the invention, reference ismade to the accompanying drawings that form a part hereof, and in whichare shown by way of illustration only of specific embodiments in whichthe invention may be practiced. It is to be understood that otherembodiments may be utilised and structural or logical changes may bemade without departing from the scope of the present invention. Thefollowing detailed description, therefore, is not to be taken in alimiting sense, and the scope of the present invention is defined by theappended claims.

Standard reference works setting forth the general principles ofrecombinant DNA technology include Molecular Cloning: A LaboratoryManual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989; Current Protocols inMolecular Biology, ed. Ausubel et al., Greene Publishing andWiley-Interscience, New York, 1992 (with periodic updates) (“Ausubel etal. 1992”); Innis et al., PCR Protocols: A Guide to Methods andApplications, Academic Press: San Diego, 1990. General principles ofmicrobiology are set forth, for example, in Davis, B. D. et al.,Microbiology, 3rd edition, Harper & Row, publishers, Philadelphia, Pa.(1980).

Disclosed herein is a gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, lacking trehalose 6-phosphatephosphorylase (TrePP) activity.

In an aspect, the gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, is for use as a medicament, i.e.,for use in treatment. A further aspect provides a medicament comprisinga gram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, lacking TrePP activity. Disclosed is also the use of agram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, lacking TrePP activity for the manufacture of amedicament. Such medicament may be provided, for example, as apharmaceutical formulation, nutraceutical, probiotic, medical orfunctional food.

Another aspect provides the use of a gram positive bacterium, preferablya lactic acid bacterium (LAB) or Bifidobacterium, lacking TrePP activityas a probiotic or a food additive, more specifically as a non-medicinalprobiotic or food additive. A related aspect provides the use of a grampositive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, lacking TrePP activity as a starter culture, preferablya starter culture for the preparation of a food product, moreparticularly wherein the food product is a non-medicinal food product.

A further aspect thus provides a probiotic or food additive, morespecifically a non-medicinal probiotic or food additive, comprising agram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, lacking TrePP activity. A related aspect provides astarter culture, preferably a starter culture for the preparation of afood product, more particularly wherein the food product is anon-medicinal food product, said starter culture comprising a grampositive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, lacking TrePP activity.

As used herein, the term “gram-positive bacterium” has its commonmeaning known in the art. By means of further guidance, a gram-positivebacterium can be identified by Gram staining as retaining crystal violetstain.

In a preferred embodiment, the gram-positive bacterium according to theinvention is non-pathogenic in the sense that it does not cause harm ordoes not lead to deleterious effects when administered to an intendedsubject.

As used herein, the term “lactic acid bacterium” of “LAB” relates to agram-positive bacterium which is non-pathogenic in the sense that itdoes not cause harm or does not lead to deleterious effects whenadministered to an intended subject, and which preferably belongs to thebacterial genera of Lactococcus, Lactobacillus, Leuconostoc,Pediococcus, Streptococcus, Aerococcus, Carnobacterium, Enterococcus,Oenococcus, Sporolactobacillus, Tetragenococcus, Vagococcus, andWeisella. More preferably, the LAB may be a Lactococcus species, suchas, but not limited to Lactococcus lactis, Lactococcus garvieae,Lactococcus piscium, Lactococcus plantarum and Lactococcusraffinolactis, and any subspecies and strains thereof. Most preferably,the Lactococcus species may be Lactococcus lactis, and any subspeciesand strain thereof, such as without limitation Lactococcus lactis ssp.cremoris, Lactococcus lactis ssp. hordniae, Lactococcus lactis ssp.lactis, Lactococcus lactis ssp. bv. diacetylactis. Further preferably,the Lactococcus lactis may be Lactococcus lactis ssp. cremoris orLactococcus lactis ssp. lactis, more preferably Lactococcus lactis ssp.cremoris, and encompasses any strains thereof, such as, e.g.,Lactococcus lactis ssp. cremoris SK11, Lactococcus lactis ssp. cremorisMG1363, or Lactococcus lactis ssp lactis IL1403. Also preferably, theLAB may an Enterococcus sp., preferably Enterococcus faecalis,Enterococcus faecium and any subspecies and strains thereof, such as,without limitation Enterococcus faecium strain LMG15709.

Bifidobacterium is a genus of Gram-positive, non-motile, often branchedanaerobic bacteria. Bifidobacteria as used herein may include B.adolescentis, B. angulatum, B. animalis, B. asteroides, B. bifidum, B.boum, B. breve, B. catenulatum, B. choerinum, B. coryneforme, B.cuniculi, B. denticolens, B. dentium, B. gallicum, B. gallinarum, B.indicum, B. infantis, B. inopinatum, B. lactis, B. longum, B. magnum, B.merycicum, B. minimum, B. pseudocatenulatum, B. pseudolongum, B.pullorum, B. ruminantium, B. saeculare, B. subtile, B. suis, B.thermacidophilum, B. thermophilum. Preferably, the Bifidobacterium is B.adolescentis, B. bifidum, B. breve, B. infantis, B. longum. It is to beunderstood that all subspecies and strains of Bifidobacteria are alsoincluded.

As used herein, the term “trehalose 6-phosphate phosphorylase”, “trePP”,or “TrePP” relates to an enzyme which phosphorylates trehalose6-phosphate, preferably an enzyme which catalyzes the reaction,preferably the reversible reaction, of α,α-trehalose-6-phosphate withphosphate to yield glucose-6-phosphate and β-D-glucose-1-phosphate, orvice versa. Synonyms for trePP are for instancetrehalose-6-phosphate:phosphate β-D-glucosyltransferase andα,α-trehalose-6-phosphate:phosphate β-D-glucosyltransferase. By means ofexample, the nucleic acid and protein sequence of trePP of Lactococcuslactis ssp. cremoris MG1363 is represented by SEQ ID NOs: 1 and 2,respectively (corresponding to Genbank accession numbers NC_009004.1(region 449195-451504) and YP_001031805.1, respectively). In anembodiment, the trePP as used herein relates to a gene or protein havingthe nucleic acid or amino acid sequence of SEQ ID NOs: 1 and 2,respectively, or having a nucleic acid encoding SEQ ID NO: 2. In afurther embodiment, the trePP as used herein relates to a gene orprotein having the nucleic acid or amino acid sequence which is at least75% identical to SEQ ID NOs: 1 and 2, respectively, such as for instanceat least 75%, 80%, 85%, 90%, 95% or more % identical. In anotherembodiment, the trePP as used herein encodes a protein which is at least75% identical to SEQ ID NO: 2, such as for instance at least 75%, 80%,85%, 90%, 95% or more % identical. Preferably, the above describedsequences relate to or encode a functional trePP protein. In anotherembodiment, the trePP as used herein is a gram positive bacterium,preferably a lactic acid bacterium (LAB) or Bifidobacterium, orthologueof SEQ ID NOs: 1 and 2.

Methods for comparing sequences and determining sequence identity arewell known in the art. By means of example, percentage of sequenceidentity refers to a percentage of identical nucleic acids or aminoacids between two sequences after alignment of these sequences.Alignments and percentages of identity can be performed and calculatedwith various different programs and algorithms known in the art.Preferred alignment algorithms include BLAST (Altschul, 1990; availablefor instance at the NCBI website) and Clustal (reviewed in Chenna, 2003;available for instance at the EBI website). Preferably, BLAST is used tocalculate the percentage of identity between two sequences, such as the“Blast 2 sequences” algorithm described by Tatusova and Madden 1999(FEMS Microbiol Lett 174: 247-250), for example using the publisheddefault settings or other suitable settings (such as, e.g., for theBLASTN algorithm: cost to open a gap=5, cost to extend a gap=2, penaltyfor a mismatch=−2, reward for a match=1, gap x_dropoff=50, expectationvalue=10.0, word size=28; or for the BLASTP algorithm: matrix=Blosum62,cost to open a gap=11, cost to extend a gap=1, expectation value=10.0,word size=3).

The activity of trePP can be measured directly or indirectly. One way toindirectly determine the activity is by means of gene sequencing. Inthis way, partial or complete deletions, disruptions or inactivatingmutations can be readily identified. A direct way to determine theactivity can for instance be based on assays with cell extracts whereinsubstrate consumption or reaction product formation is measured (e.g.the substrate trehalose 6-phosphate or the reaction productsglucose-6-phosphate and β-D-glucose-1-phosphate), possibly combined withprior metabolic labelling. Substrate and products can also be readilydetermined by for instance high performance anion exchangechromatography (HPAEC), as for instance described in Andersson et al.2001 (supra).

As used herein, the term “lacking trePP activity” means that no orsubstantially no trePP activity is present. By means of furtherguidance, the trePP activity is less than 20% of the trePP activity ofwild type gram positive bacterium, preferably a lactic acid bacterium(LAB) or Bifidobacterium. For instance, the trePP activity is less than15%, preferably less than 10%, more preferably less than 5%, even morepreferably less than 1% of wild type trePP activity. As indicatedbefore, most preferably the trePP activity is undetectable orsubstantially or completely absent.

As used herein, the term “medicament” also encompasses the terms “drug”,“therapeutic”, and other terms which are used in the field of medicineto indicate a preparation with therapeutic or prophylactic effect.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder. The terms “treatment”, “treating”, andthe like, as used herein also include amelioration or elimination of adeveloped disease or condition once it has been established oralleviation of the characteristic symptoms of such disease or condition.As used herein these terms also encompass, depending on the condition ofthe patient, preventing the onset of a disease or condition or ofsymptoms associated with a disease or condition, including reducing theseverity of a disease or condition or symptoms associated therewithprior to affliction with said disease or condition. Such prevention orreduction prior to affliction refers to administration of the compoundor composition of the invention to a patient that is not at the time ofadministration afflicted with the disease or condition. “Preventing”also encompasses preventing the recurrence or relapse-prevention of adisease or condition or of symptoms associated therewith, for instanceafter a period of improvement.

As used herein, “nutraceuticals” generally encompass foods or foodproducts that provide health and medical benefits. Nutraceuticals areedible and may be eaten directly by humans, but are preferably providedto humans in the form of additives or nutritional supplements, e.g., inthe form of tablets of the kind sold in health food stores, or asingredients in edible solids, more preferably processed food productssuch as cereals, breads, tofu, cookies, ice cream, cakes, potato chips,pretzels, cheese, etc., and in drinkable liquids e.g., beverages such asmilk, soda, sports drinks, and fruit juices. Especially preferredprocesses for producing nutraceuticals involve only naturally derivedsolvents. Nutraceuticals may preferably contain relatively high levelsof health-enhancing substances Nutraceuticals may be intermixed with oneanother to increase their health-enhancing effects.

In contrast to nutraceuticals, the so-called “medical foods” are notmeant to be used by the general public and are not available in storesor supermarkets. Medical foods are not those foods included within ahealthy diet to decrease the risk of disease, such as reduced-fat foodsor low-sodium foods, nor are they weight loss products. A physicianprescribes a medical food when a patient has special nutrient needs inorder to manage a disease or health condition, and the patient is underthe physician's ongoing care. The label states that the product isintended to be used to manage a specific medical disorder or condition.An example of a medical food is nutritionally diverse medical fooddesigned to provide targeted nutritional support for patients withchronic inflammatory conditions. Active compounds of this product arefor instance one or more of the compounds described herein. Functionalfoods may encompass those foods included within a healthy diet todecrease the risk of disease, such as reduced-fat foods or low-sodiumfoods, or weight loss products.

As used herein, the term “probiotics” refers to bacteria that helpmaintain the natural balance of microorganisms (microflora) in theintestines camera. Also, the normal human digestive tract containsprobiotic bacteria that reduce the growth of harmful bacteria andpromote a healthy digestive system. The largest group of probioticbacteria in the intestine is LAB. As used herein, a “probioticcomposition” is a composition, preferably an edible composition,comprising a probiotic. The term “probiotic composition” as used hereinmay be used interchangeably with “dietary supplement”. The probioticcomposition as defined herein can find use as supplement to food andbeverages, and as pharmaceutical formulations for enteral or parenteralapplication which may be solid formulations such as capsules or tablets,or liquid formulations, such as solutions or suspensions. Suchformulations may include without limitation drinks (e.g. Actimel®,Yakult®, DanActive® . . . ), drink yoghurts, yoghurt, fresh cheese,cream, sour cream, etc. Hence, it shall be appreciated that a probioticor probiotic composition may be for medicinal or non-medicinalapplications.

The term “starter culture” refers to a microbiological culture whichactually performs fermentation. These starters usually consist of acultivation medium, such as grains, seeds, or nutrient liquids that havebeen well colonized by the microorganisms used for the fermentation. Asused herein, the term starter culture preferably refers to a highdensity starter culture. Accordingly, a starter culture may refer to acomposition comprising live microorganisms that are capable ofinitiating or effecting fermentation of organic material, optionallyafter being cultivated in a separate starter medium for obtaining a highdensity culture. Alternatively, the starter culture may be dried,spray-dried, frozen or freeze-dried.

As indicated before, the present inventors have surprisingly found thatthe mere absence of trePP is sufficient for intracellular trehaloseaccumulation in gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium. In contrast herewith, it has beenpreviously thought that the presence of a heterologous trehalose6-phosphate phosphatase and/or a heterologous trehalose 6-phosphatesynthase, such as otsB and otsA, respectively, is essential forintracellular trehalose accumulation. Accordingly, in an embodiment, theinvention relates to the gram positive bacterium, preferably a lacticacid bacterium (LAB) or Bifidobacterium, as described herein, notcontaining functional heterologous otsB, preferably no functionalheterologous trehalose 6-phosphate phosphatase. In a further embodiment,the gram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, as described herein does not contain a functionalheterologous otsA, preferably no functional heterologous trehalose6-phosphate synthase. In yet another embodiment, the gram positivebacterium, preferably a lactic acid bacterium (LAB) or Bifidobacterium,as described herein does not contain a functional heterologous otsA anddoes not contain a functional heterologous otsB. In a furtherembodiment, the gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, as described herein does not containa functional heterologous trehalose 6-phosphate synthase and does notcontain a functional heterologous trehalose 6-phosphate phosphatase. Inyet a further embodiment, the gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, as described herein doesnot contain a functional heterologous gene involved in the metabolism(either catabolic or anabolic) of trehalose or trehalose 6-phosphate.Such genes encompass, without limitation trehalase, trehalosephosphorylase, and trehalose 6-phosphate hydrolase.

As used herein, the term “does not contain” or “not containing”preferably relates to gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, which do not express a particulargene product, i.e. no functional or active protein is produced in saidgram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium.

As used herein, the term “trehalose 6-phosphate phosphatase” relates toan enzyme which dephosphorylates trehalose 6-phosphate, preferably anenzyme which catalyzes the reaction of trehalose-6-phosphate to yieldphosphate and trehalose. Trehalose 6-phosphate phosphatase belongs tothe family of Phosphoric Monoester Hydrolases. Synonyms for trehalose6-phosphate phosphatase are for instance α,α-trehalose-6-phosphatephosphohydrolase, trehalose-6-phosphate phosphohydrolase, and trehalose6-phosphatase. By means of example, the nucleic acid and proteinsequence of trehalose 6-phosphate phosphatase of E. coli (i.e., otsB) isrepresented by SEQ ID NOs: 3 and 4, respectively (corresponding toGenbank accession numbers X69160.1 (nucleotide positions 675-1475) andP31678.2, respectively). In an embodiment, the trehalose 6-phosphatephosphatase as used herein relates to a gene or protein having thenucleic acid or amino acid sequence of SEQ ID NOs: 3 and 4,respectively, or having a nucleic acid encoding SEQ ID NO: 4. In afurther embodiment, the trehalose 6-phosphate phosphatase as used hereinrelates to a gene or protein having the nucleic acid or amino acidsequence which is at least 75% identical to SEQ ID NOs: 3 and 4,respectively, such as for instance at least 75%, 80%, 85%, 90%, 95% ormore % identical. In another embodiment, the trehalose 6-phosphatephosphatase as used herein encodes a protein which is at least 75%identical to SEQ ID NO: 4, such as for instance at least 75%, 80%, 85%,90%, 95% or more % identical. Preferably, the above described sequencesrelate to or encode a functional trehalose 6-phosphate phosphataseprotein. In another embodiment, the trehalose 6-phosphate phosphatase asused herein is a gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, orthologue of SEQ ID NOs: 3 and 4.

As used herein, the term “trehalose 6-phosphate synthase” relates to anenzyme which dephosphorylates trehalose 6-phosphate, preferably anenzyme which catalyzes the reaction of glucose 6-phosphate withUDP-glucose to yield trehalose 6-phosphate. Trehalose 6-phosphatesynthase belongs to the family of glycosyltransferases. Synonyms fortrehalose 6-phosphate synthase are for instance trehalosephosphate-uridine diphosphate glucosyltransferase,phosphotrehalose-uridine diphosphate transglucosylase, uridinediphosphoglucose phosphate glucosyltransferase, andα,α-trehalose-6-phosphate synthase. By means of example, the nucleicacid and protein sequence of trehalose 6-phosphate synthase of E. coliotsA) is represented by SEQ ID NOs: 5 and 6, respectively (correspondingto Genbank accession numbers X69160.1 (nucleotide positions 1450-2874)and P31677.3, respectively). In an embodiment, the trehalose 6-phosphatesynthase as used herein relates to a gene or protein having the nucleicacid or amino acid sequence of SEQ ID NOs: 5 and 6, respectively, orhaving a nucleic acid encoding SEQ ID NO: 6. In a further embodiment,the trehalose 6-phosphate synthase as used herein relates to a gene orprotein having the nucleic acid or amino acid sequence which is at least75% identical to SEQ ID NOs: 5 and 6, respectively, such as for instanceat least 75%, 80%, 85%, 90%, 95% or more % identical. In anotherembodiment, the trehalose 6-phosphate synthase as used herein encodes aprotein which is at least 75% identical to SEQ ID NO: 6, such as forinstance at least 75%, 80%, 85%, 90%, 95% or more % identical.Preferably, the above described sequences relate to or encode afunctional trehalose 6-phosphate synthase protein. In anotherembodiment, the trehalose 6-phosphate synthase as used herein is a grampositive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, orthologue of SEQ ID NOs: 5 and 6.

It is to be understood that, as used herein, the absence of functionalheterologous trehalose 6-phosphate phosphatase or synthase (or any othertrehalose related metabolic enzyme) relates to the absence of any orsubstantially any heterologous trehalose 6-phosphate phosphatase orsynthase activity. Such activity can be determined as described hereinelsewhere. In an embodiment, the gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, as described herein donot express these enzymes.

The inventors have found that trehalose to some extent leaks from cellsthrough an up to now unidentified or unanticipated trehalose exit portand can be recovered in the supernatant. Surprisingly, the inventorsfound that the disruption of ptcC circumvents the release of trehalose.Accordingly, in an embodiment, the invention relates to gram positivebacterium, preferably a lactic acid bacterium (LAB) or Bifidobacterium,as described herein, lacking cellobiose-specific PTS system IICcomponent (ptcC) activity.

“Cellobiose-specific PTS system IIC component” or “ptcC” as used hereinrefers to a phosphotransferase system component. The phosphotransferasesystem is involved in catalyzing the transfer of the phosphoryl groupfrom phosphoenolpyruvate to incoming sugar substrates concomitant withtheir translocation across the cell membrane. ptcC is the transmembranecomponent of a cellobiose-specific PTS system. ptcC has up till now notbeen implicated in trehalose transport, let alone being involved intrehalose leakage from gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium. By means of example, the nucleicacid and protein sequence of ptcC of Lactococcus lactis ssp. cremorisMG1363 is represented by SEQ ID NOs: 7 and 8, respectively(corresponding to Genbank accession numbers NC_009004.1 (region430271-431608) and YP_001031790.1, respectively). In an embodiment, theptcC as used herein relates to a gene or protein having the nucleic acidor amino acid sequence of SEQ ID NOs: 7 and 8, respectively, or having anucleic acid encoding SEQ ID NO: 8. In a further embodiment, the ptcC asused herein relates to a gene or protein having the nucleic acid oramino acid sequence which is at least 75% identical to SEQ ID NOs: 7 and8, respectively, such as for instance at least 75%, 80%, 85%, 90%, 95%or more % identical. In another embodiment, the ptcC as used hereinencodes a protein which is at least 75% identical to SEQ ID NO: 8, suchas for instance at least 75%, 80%, 85%, 90%, 95% or more % identical.Preferably, the above described sequences relate to or encode afunctional ptcC protein. In another embodiment, the ptcC as used hereinis a gram positive bacterium, preferably a lactic acid bacterium (LAB)or Bifidobacterium, orthologue of SEQ ID NOs: 7 and 8.

The gram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, lacking trePP and/or ptcC activity according to theinvention can be obtained by any means known in the art, be it usingmolecular biological methodology or obtained through high throughputscreening of natural variants or variants obtained from random chemicalor irradiation mutagenesis. (High throughput screening for trePP KO canbe performed by a method using the absence of growth on trehalose of thetrePP defective strain or by high throughput sequencing andbioinformatic analysis of trePP orthologs or other methods). (forbackground relating to recombinant techniques and genetic manipulationof LAB see for instance “Genetics and Biotechnology of Lactic AcidBacteria”, eds. Gasson & de Vos, Blackie Academic & Professional, 1994and “Genetics of Lactic Acid Bacteria”, eds. Wood & Warner, Springer,2003) In an embodiment, in the gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, according to theinvention the gene encoding endogenous TrePP and/or PtcC and/or thepromoters from which trePP and/or ptcC are expressed has been partiallyor completely deleted, disrupted or inactivated such as being incapableof producing functional trePP and/or ptcC gene product. Techniques forgene disruption are generally known in the art. By means of example, theendogenous trePP and/or ptcC gene can be inactivated by complete orpartial removal of the coding region (knock-out) or alternativelycomplete or partial removal or mutagenesis of the promoter region.Alternatively, the trePP and/or ptcC gene may be insertionallyinactivated (knock-in), thereby disrupting the endogenous codingsequence. For instance, premature stop codons or frame shift mutationsmay be introduced. The trePP and/or ptcC gene may also be mutagenized byintroduction of one or more missense or nonsense mutations, as long asno or substantially no functional trePP and/or ptcC protein can beproduced anymore, i.e. trePP and/or ptcC activity is (substantially)absent. It is to be understood that spontaneous mutations are alsocovered.

The inventors have further found that overexpressing one or moretrehalose transporters further augments intracellular trehaloseaccumulation and/or retention in gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium.

Accordingly, in an embodiment, the invention relates to gram positivebacterium, preferably a lactic acid bacterium (LAB) or Bifidobacterium,as described herein, overexpressing, preferably constitutivelyoverexpressing, one or more genes encoding a trehalose transporter. In apreferred embodiment, said trehalose transporters are endogenoustrehalose transporters of gram positive bacterium, preferably a lacticacid bacterium (LAB) or Bifidobacterium. In a further preferredembodiment, the trehalose transporters are endogenous trehalosetransporters located in the trehalose operon of gram positive bacterium,preferably a lactic acid bacterium (LAB) or Bifidobacterium. In yetanother embodiment, the trehalose transporters are endogenous trehalosetransporters of the phosphotransferase system (PTS) located within thetrehalose operon of gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium. In a preferred embodiment, theoverexpression of the one or more trehalose transporters as describedherein is accomplished by insertion of a promoter 5′ to the one or moretransporters such that the promoter is operably linked to thetransporter sequence(s). Operably linked refers to a juxtapositionwherein the components so described are in a relationship permittingthem to function in their intended manner. A promoter sequence “operablylinked” to a coding sequence is ligated in such a way that expression ofthe coding sequence is achieved under conditions compatible with thepromoter sequence. In an embodiment, said promoter is a strong promoter.In a further embodiment, said promoter is a constitutive promoter. Inyet another embodiment, said promoter is an endogenous gram positivebacterium, preferably a lactic acid bacterium (LAB) or Bifidobacterium,promoter. Suitable promoters can be found for instance in WO2008/084115, which incorporated herein in its entirety. In particular,the promoters listed in Table 12 of WO 2008/084115 are particularlysuited to overexpress the transporters as described herein. Mostpreferably, the promoter is PhIIA (i.e. the promoter of the HU-likeDNA-binding protein). Accordingly, in a preferred embodiment the PhIIApromoter is inserted upstream of the coding regions of the endogenoustrehalose transporter(s) located in the trehalose operon of grampositive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium. In an embodiment, the PhIIA promoter has the sequenceof SEQ ID NO: 13, corresponding to the PhIIA promoter of Lactococcuslactis ssp. cremoris MG1363. In another embodiment, the PhIIA promoterhas a sequence which is at least 75% identical to SEQ ID NO: 13, such asat least 75%, 80%, 85%, 90%, 95% or more identical to SEQ ID NO: 13. Ina further embodiment, the PhIIA is a gram positive bacterium, preferablya lactic acid bacterium (LAB) or Bifidobacterium, orthologue of SEQ IDNO: 13.

By means of example, the trehalose transporters referred to herein arerepresented by the Lactococcus lactis ssp. cremoris MG1363 nucleic acidand amino acid sequence of SEQ ID NOs: 9 and 10, respectively(corresponding to Genbank accession numbers NC_009004.1 (region446937-447422) and YP_001031803.1, respectively), and/or SEQ ID NOs: 11and 12, respectively (corresponding to Genbank accession numbersNC_009004.1 (region 447563-449128) and YP_001031804.1, respectively). Inan embodiment, the overexpressed transporter(s) as used herein relate toa gene or protein having the nucleic acid or amino acid sequence of SEQID NOs: 9 and 10, respectively, and/or SEQ ID NOs: 11 and 12,respectively, or having a nucleic acid encoding SEQ ID NO: 10 and/or SEQID NO: 12. In a further embodiment, the overexpressed transporter(s) asused herein relates to a gene or protein having the nucleic acid oramino acid sequence which is at least 75% identical to SEQ ID NOs: 9 and10, respectively, and/or SEQ ID NOs: 11 and 12, respectively, such asfor instance at least 75%, 80%, 85%, 90%, 95% or more % identical. Inanother embodiment, the overexpressed transporter(s) as used hereinencodes a protein which is at least 75% identical to SEQ ID NO: 10and/or SEQ ID NO: 12, such as for instance at least 75%, 80%, 85%, 90%,95% or more % identical. Preferably, the above described sequencesrelate to or encode (a) functional overexpressed, preferablyconstitutively overexpressed, transporter(s) protein(s). In anotherembodiment, the (constitutively) overexpressed transporter(s) as usedherein is(are) a gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, orthologue(s) of SEQ ID NOs: 9 and10 and/or SEQ ID NOs: 11 and 12.

In an embodiment, the invention relates to gram positive bacterium,preferably a lactic acid bacterium (LAB) or Bifidobacterium, which lacktrePP and further displaying any one or preferably both of the followingcharacteristics: (i) the gram positive bacterium lacks ptcC activity;(ii) the gram positive bacterium overexpresses, preferablyconstitutively overexpresses, one or more trehalose transporters,wherein the gram positive bacterium in addition contains functionalheterologous trehalose 6-phosphate phosphatase, such as otsB and/orfunctional heterologous trehalose 6-phosphate synthase, such as otsA,more preferably wherein the gram positive bacterium contains at leastsaid functional heterologous trehalose 6-phosphate phosphatase, evenmore preferably wherein the gram positive bacterium contains saidfunctional heterologous trehalose 6-phosphate phosphatase but does notcontain said functional heterologous trehalose 6-phosphate synthase.Each of these proteins is as described herein elsewhere. Particularlypreferred is a genomic integration of the trehalose 6-phosphatephosphatase, wherein the integration is preferably as disclosed inEuropean patent applications with application numbers 11168495.7 and11173588.2. These applications relate to dual cistron expression systemsand are incorporated herein by reference in their entirety. Thepreferred position of trehalose 6-phosphate phosphatase, preferablyotsB, as it is used here, is as a second cistron behind the endogenoususp45 gene.

The gram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, as described herein show an increased tolerance towardsvarious environmental and storage associated insults or stress, such asan increased drying, spray-drying, freezing or freeze-drying resistance,as well as an increased resistance towards the harsh conditions in thegastrointestinal tract (e.g. acids and bile salts). The gram positivebacterium, preferably a lactic acid bacterium (LAB) or Bifidobacterium,according to the invention are therefore particularly well suited to beadministered to a subject while showing an increased survival rate inthe gastrointestinal tract. These gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, can therefore also beapplied to deliver proteins to a subject. Accordingly, in an embodiment,the invention relates to gram positive bacterium, preferably a lacticacid bacterium (LAB) or Bifidobacterium, as described herein, containingone or more heterologous gene product, preferably one or moreprophylactic and/or therapeutic gene product and/or antigen. Delivery ofbiological active polypeptides has for instance been described in WO97/14806, WO 00/23471, WO 01/02570, WO 02/090551, WO 2005/111194, WO2007/025977, WO 2007/063075, WO 2007/128757, WO 2008/071751, WO2008/090223, WO 2004/046346, and WO 2010/034844. Preferably, theheterologous genes as described herein are integrated into the bacterialgenome. A particularly preferred integration strategy is disclosed inEuropean patent applications with application numbers 11168495.7 and11173588.2, which are incorporated herein in their entirety byreference. In particular, the heterologous genes may be insertedpolycistronically (e.g., bi-, tri- or multi-cistronically) as a second(or further) gene in a native (endogenous) locus, preferably an operon.In this way, the heterologous gene is expressed under control of anative (endogenous) promoter.

As used herein, the term “antigen” generally refers to a substance thatevokes an immune response, including humoral immunity and/or cellularimmunity response, and that is capable of binding with a product, e.g.,an antibody or a T cell, of the immune response. An antigen as intendedherein may in an alternative be such as to induce immuno-tolerance,e.g., may be an auto-antigen (including auto- and allo-antigens) or maybe allergen. Hence, in a preferred example, an antigen requires afunctioning immune system of a subject to which it is administered toelicit a physiological response from such a subject. The “antigen” asintended herein also encompasses “self-antigens” which do not provoke animmune response in a healthy individual but would do so in a personsuffering from auto-immune disease, i.e. the failure of an organism torecognize its own constituent parts (down to the sub-molecular levels)as “self”, which results in an immune response against its own cells andtissues. Any disease that results from such an aberrant immune responseis termed an autoimmune disease. Accordingly, the “antigen” as intendedherein also encompasses a (physiologically active) protein which wouldnot provoke an immune response in a healthy individual but would do soin a person genetically deficient in said protein. In addition, the“antigen” as intended herein also encompasses an allergen which wouldnot provoke an immune response in a healthy individual but would do soin a person suffering from an allergic disease.

An antigen as intended herein may be derived from any polypeptide towhich an immune response in a human or animal subject would betherapeutically useful, e.g., from a pathogen, e.g., from a viral,prokaryotic (e.g., bacterial) or eukaryotic pathogen, from anon-physiological protein (e.g., a protein derived from cancer tissue),from allergen (e.g., for eliciting immune tolerance), etc. An antigencould also be a metabolite of a protein. As an example, the antigencould be a polysaccharide, a lipid or other. Strong promoters asdescribed here could drive the expression of the necessary enzymes tosynthesize or assemble said polysaccharide, lipid or other.

The term “a prophylactically and/or therapeutically gene product”,polypeptide or protein refers generally to a peptide, polypeptide orprotein that, in a human or animal subject to which it is administered,does not elicit an immune response against itself (i.e., isnon-vaccinogenic) and is able to achieve a prophylactic and/ortherapeutic effect. Hence, the prophylactic and/or therapeutic effect ofsuch a peptide, polypeptide or protein would be expected to be directlylinked to its own natural biological function whereby it can achieveparticular effects in a body of a subject; rather than producing aprophylactic and/or therapeutic effect by acting as an immunogenicand/or immunoprotective antigen in the subject. Hence, thenon-vaccinogenic prophylactically and/or therapeutically active peptide,polypeptide or protein should be biologically active in its expressedform or, at least, must be converted into the biologically active formonce released from the expressing host cell. Preferably, suchbiologically active form of the said peptide, polypeptide or protein maydisplay a secondary and preferably also tertiary conformation which isthe same or closely analogous to its native configuration.

Preferably, the prophylactic and/or therapeutic gene product,polypeptide or protein is also non-toxic and non-pathogenic. In apreferred embodiment, the prophylactically and/or therapeutically geneproduct, polypeptide or protein may be derived from human or animal, andmay preferably correspond to the same taxon as the human or animalsubject to which it is to be administered.

Non-limiting examples of suitable prophylactically and/ortherapeutically gene products, polypeptides or proteins include oneswhich are capable of functioning locally or systemically, e.g., is/arecapable of exerting endocrine activities affecting local or whole-bodymetabolism and/or is/are capable of the regulation of the activities ofcells belonging to the immunohaemopoeitic system and/or is/are capableof affecting the viability, growth and differentiation of a variety ofnormal or neoplastic cells in the body or affecting the immuneregulation or induction of acute phase inflammatory responses to injuryand infection and/or is/are capable of enhancing or inducing resistanceto infection of cells and tissues mediated by chemokines acting on theirtarget cell receptors, or the proliferation of epithelial cells or thepromotion of wound healing and/or is/are capable of modulating theexpression or production of substances by cells in the body. Specificexamples of such peptides, polypeptides and proteins include, withoutlimitation, insulin, growth hormone, prolactin, calcitonin, luteinisinghormone, parathyroid hormone, somatostatin, thyroid stimulating hormone,vasoactive intestinal polypeptide, cytokines such as IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, any of IL-14 toIL-32, in particular IL-27, GM-CSF, M-CSF, SCF, IFNs, EPO, G-CSF, LIF,OSM, CNTF, GH, PRL, the TNF family of cytokines, e.g., TNFα, TNFα, CD40,CD27 or FAS ligands, the IL-1 family of cytokines, the fibroblast growthfactor family, the platelet derived growth factors, transforming growthfactors and nerve growth factors, the epidermal growth factor family ofcytokines, the insulin related cytokines, etc. Alternatively, theprophylactically and/or therapeutically active polypeptide can be areceptor or antagonist for the prophylactically and/or therapeuticallyactive polypeptides as defined above. Alternatively, theprophylactically and/or therapeutically active polypeptide can be anantibody, such as a neutralizing antibody, or the likes thereof. Furtherspecific examples of such suitable polypeptides are listed, e.g., in WO96/11277, page 14, lines 1-30, incorporated herein by reference; in WO97/14806, page 12, line 1 through page 13, line 27, incorporated hereinby reference; or U.S. Pat. No. 5,559,007, col. 8, line 31 through col.9, line 9, incorporated by reference herein. In an example, saidprophylactically and/or therapeutically active peptide, polypeptide orprotein may be IL-10, more preferably hIL-10, glucagon-like peptide-1(GLP-1), more preferably hGLP-1, glucagon-like peptide-2 (GLP-2), morepreferably hGLP-2, trefoil factors (TFF, e.g., TFF1, 2 and/or 3), orPYY, more preferably hPYY.

As mentioned, in embodiments the prophylactically and/or therapeuticallyactive polypeptide can be an antibody, such as a neutralizing antibody,or the likes thereof. The antibody as described herein can be a fullsize antibody or a functional fragment thereof such as Fab, a fusionprotein or a multimeric protein. In a preferred embodiment, the one ormore heterologous genes encodes an antibody or a functional antibodyfragment. As used herein, the term “functional” refers to an antibodyfragment, which can still exert its intended function, i.e. antigenbinding. The term antibody, as used here, includes, but is not limitedto conventional antibodies, chimeric antibodies, dAb, bispecificantibody, trispecific antibody, multispecific antibody, bivalentantibody, trivalent antibody, multivalent antibody, VHH, nanobody, Fab,Fab′, F(ab′)₂ scFv, Fv, dAb, Fd, diabody, triabody, single chainantibody, single domain antibody, single antibody variable domain.

In the present context, the term “antibody” is used to describe animmunoglobulin whether natural or partly or wholly engineered. Asantibodies can be modified in a number of ways, the term “antibody”should be construed as covering any specific binding molecule orsubstance having a binding domain with the required binding specificityfor the other member of the pair of molecules, i.e. the target molecule,as defined supra. Thus, this term covers antibody fragments,derivatives, functional equivalents and homologues of antibodies, aswell as single chain antibodies, bifunctional antibodies, bivalentantibodies, VHH, nanobodies, Fab, Fab′, F(ab′)₂, scFv, Fv, dAb, Fd,diabodies, triabodies and camelid antibodies, including any polypeptidecomprising an immunoglobulin binding domain, whether natural or whollyor partially engineered. Chimeric molecules comprising an immunoglobulinbinding domain, or equivalent, fused to another polypeptide aretherefore included. The term also covers any polypeptide or proteinhaving a binding domain which is, or is homologous to, an antibodybinding domain, e.g. antibody mimics. Examples of antibodies are theimmunoglobulin isotypes and their isotypic subclasses, including IgG(IgG1, IgG2a, IgG2b, IgG3, IgG4), IgA, IgD, IgM and IgE. The person inthe art will thus appreciate that the present invention also relates toantibody fragments, comprising an antigen binding domain such as VHH,nanobodies Fab, scFv, Fv, dAb, Fd, diabodies and triabodies. In anembodiment, the invention relates to a gram-positive bacterium or arecombinant nucleic acid as described herein, wherein one exogenous geneencodes the light chain (V_(L)) of an antibody or of a functionalfragment thereof, and another exogenous gene encodes the heavy chain(V_(H)) of the antibody or of a functional fragment thereof, morepreferably wherein the functional fragment is Fab. In an embodiment, theexogenous gene encoding V_(L) or functional fragment thereof istranscriptionally coupled to the 3′ end of the exogenous gene encodingV_(H) or functional fragment thereof.

In an embodiment, the (neutralizing) antibody as described herein atleast partially or fully blocks, inhibits, or neutralises a biologicalactivity of a target molecule, such as a cytokine or chemokine or atoxin. As used herein, the expression “neutralises” or “neutralisation”means the inhibition of or reduction in a biological activity of acytokine or toxin as measured in vivo or in vitro, by methods known inthe art, such as, for instance, as detailed in the examples. Inparticular, the inhibition or reduction may be measured by determiningthe colitic score or by determining the target molecule in a tissue orblood sample. As used herein, the expression “neutralises” or“neutralisation” means the inhibition of or reduction in a biologicalactivity of a cytokine or toxin as measured in vivo or in vitro, by atleast 10% or more, preferably by at least 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% and even more preferably by 100%.

Preferably, said antibody or functional fragment thereof inhibit thebiological effect of cytokines chosen from the list of IL-1β, IL-2,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12 (or its subunits IL-12p35 andIL12p40), IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23 (or itssubunit IL-23p19), IL-27, IL-32 (and its splice variants), IFN (α, β, γ)and TNFα. Alternatively, these cytokines may be inhibited by bindingmolecules which are not antibodies. Preferably, said binding moleculesare soluble cytokine receptors such as gp130, or are binding to thereceptors of said cytokines, for example IL-2R (CD25, CD122, CD132),IL-12R (beta1, beta2), IL15R, IL-17R, IL-23R or IL-6R, withouttriggering an inflammatory signal. Preferably, said binding moleculesare neutralizing chemokines chosen from the list of MIF, MIP-1α, MCP-1,RANTES and Eotaxin. Preferably, said binding molecules are solving theblockade of immune activation via binding to costimulatory moleculesfrom the list of CD3/CD28, HVEM, B7.1/B7.2, CD40/CD40L(CD154),ICOS/ICOSL, OX40/X40L, CD27/CD27L(CD70), CD30/CD30L(CD153) and 41 BB/41BBL. Preferably, said binding molecules are solving the blockade ofinflammation via binding to adhesion molecules from the list I-CAM1, α4integrin and α4β7 integrin. Preferably, said binding molecules have acostimulatory and agonistic effect on CD3, CTLA4 and/or PD1. Preferably,said binding molecules are neutralizing T-cells or B-cell activity bytargeting CD25, CD20, CD52, CD95, BAFF, APRIL and/or IgE. Preferably,said binding molecules are solving the blockade of inflammation viabinding to enzymes from the MMP family. Preferably, said bindingmolecules assert an anti-angiogenic effect, such as neutralizingαvβ3/α5β1 and IL-8 activity. In a further preferred embodiment saidbinding molecule or antibody (or functional fragment) is capable ofneutralizing the biological effect of TNFα, IL-12, IFNγ, IL-23 or IL-17.

Non-limiting examples of antibodies or binding molecules which can beused as heterologous genes in the gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, as described hereininclude:

-   -   an anti-TNFα antibody, anti-TNFα antibody fragment, anti-TNFα        single antibody variable domain, soluble TNF receptor or        dominant negative variant of TNFα;    -   anti-IL-12 antibody, anti-IL-12 antibody fragment, anti-IL-12        single antibody variable domain, soluble IL-12 receptor,        dominant negative variant of IL-12 or IL-12 dAb;    -   anti-IL-12p35 antibody, anti-IL-12p35 antibody fragment,        anti-IL-12p35 single antibody variable domain, soluble IL-12p35        receptor, dominant negative variant of IL-12p35 or IL-12p35 dAb;

anti-IL-12p40 antibody, anti-IL-12p40 antibody fragment, anti-IL-12p40single antibody variable domain, soluble IL-12p40 receptor, dominantnegative variant of IL-12p40 or IL-12p40 dAb;

anti-IL-23 antibody, anti-IL-23 antibody fragment, anti-IL-23 singleantibody variable domain, soluble IL-23 receptor, dominant negativevariant of IL-23 or IL-23 dAb;

anti-IL-23p19 antibody, anti-IL-23p19 antibody fragment, anti-IL-23p19single antibody variable domain, soluble IL-23p19 receptor, dominantnegative variant of IL-23p19 or IL-23p19 dAb;

-   -   an anti-IFNγ antibody, anti-IFNγ antibody fragment, anti-IFNγ        single antibody variable domain, soluble IFNγ receptor or        dominant negative variant of IFNγ;

anti-IL-17 antibody, anti-IL-17 antibody fragment, anti-IL-17 singleantibody variable domain, soluble IL-17 receptor, dominant negativevariant of IL-17 or IL-17 dAb; and

-   -   anti-MCP-1 antibody, anti-MCP-1 antibody fragment, anti-MCP-1        single antibody variable domain, soluble IL-17 receptor,        dominant negative variant of MCP-1 or MCP-1 dAb.

In a preferred embodiment, said antibody is a Fab fragment (fragmentantigen-binding). Fab fragments are well known in the art. By means offurther guidance, a Fab fragment is a region on an antibody that bindsto antigens. It is composed of one constant and one variable domain ofeach of the heavy and the light chain.

In an embodiment, the Fab is cA2 anti-TNF Fab (of which thepolynucleotide and polypeptide sequences of the variable domain of theheavy chain and the light chain are disclosed in U.S. Pat. No. 6,790,444as SEQ ID NO: 4 and 5 (heavy chain) and SEQ ID NO: 2 and 3 (lightchain), respectively) or CDP870 anti-TNF Fab (of which thepolynucleotide and polypeptide sequences of the heavy chain and thelight chain are disclosed in WO 01/94585 as SEQ ID NO: 114 and 115(heavy chain) and SEQ ID NO: 112 and 113 (light chain), respectively).

The skilled person will appreciate that antibodies, as are functionalantibody fragments, and in particular Fab fragments, are composed ofdifferent individual polypeptides which may be covalently linked bydisulphide bridges. In particular, the heavy chain and the light chainare encoded by separate individual coding sequences.

Accordingly, in an embodiment the heterologous gene disclosed hereinencodes an antigen and/or a (neutralizing) antibody or functionalfragment or variant thereof and/or a prophylactically and/ortherapeutically active peptide, polypeptide or protein, wherein the saidantigen is capable of eliciting an immune response, preferablyprotective immune response or immune tolerance response, in a human oranimal subject, and/or the said prophylactically and/or therapeuticallygene product, polypeptide or protein is capable of producing aprophylactic and/or therapeutic effect in a human or animal subject.

In an embodiment, the invention relates to gram positive bacterium,preferably a lactic acid bacterium (LAB) or Bifidobacterium, asdescribed herein or the gram positive bacterium, preferably a lacticacid bacterium (LAB) or Bifidobacterium, for use as described hereinwhich are formulated for storage. In particular, in an embodiment, theinvention relates to gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, as described herein which arefrozen, dried, freeze-dried, spray-dried or stored in medium.

As explained heretofore, the invention also relates to compositionscomprising the gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, as described herein or comprisingthe gram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, for use as described herein. Such composition may be apharmaceutical composition. In a further embodiment, the inventionrelates to a composition or a pharmaceutical composition for use intreatment or for use as a medicament, a nutraceutical, a medical food, afunctional food, a probiotic composition, a food additive or a starterculture. In yet another embodiment, the invention relates to the use ofsuch composition or pharmaceutical composition as a medicament,nutraceutical, medical food, functional food, probiotic, food additive,starter culture, or for the preparation of a medicament, nutraceutical,medical food, functional food, probiotic composition, food additive,starter culture.

As used herein, the medicinal compositions as described herein, such aspharmaceutical formulation, nutraceutical, medical or functional food orprobiotic, preferably comprises a therapeutically effective amount ofthe gram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, of the invention and a pharmaceutically acceptablecarrier, i.e., one or more pharmaceutically acceptable carriersubstances and/or additives, e.g., buffers, carriers, excipients,stabilisers, etc. The term “pharmaceutically acceptable” as used hereinis consistent with the art and means compatible with the otheringredients of a pharmaceutical composition and not deleterious to therecipient thereof.

In an embodiment, the pharmaceutical composition comprises atherapeutically effective amount of the gram positive bacterium,preferably a lactic acid bacterium (LAB) or Bifidobacterium, asdescribed herein. The term “therapeutically effective amount” refers toan amount of a therapeutic substance or composition effective to treat adisease or disorder in a subject, e.g., human or animal, i.e., to obtaina desired local or systemic effect and performance. By means of example,a therapeutically effective amount of bacteria may comprise at least 1bacterium, or at least 10 bacteria, or at least 10² bacteria, or atleast 10³ bacteria, or at least 10⁴ bacteria, or at least 10⁶ bacteria,or at least 10⁶ bacteria, or at least 10⁷ bacteria, or at least 10⁸bacteria, or at least 10⁹, or at least 10¹⁰, or at least 10¹¹, or atleast 10¹², or at least 10¹³, or at least 10¹⁴, or at least 10¹⁵, ormore host cells, e.g., bacteria, e.g., in a single or repeated dose. Thegram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, of the present invention may be administered alone orin combination with one or more active compounds. The latter can beadministered before, after or simultaneously with the administration ofthe gram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, according to the invention.

Preferably the gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, as described herein or compositioncomprising these gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, is provided in a unit dosage form,for example a tablet, capsule, enema or metered aerosol dose, so that asingle dose is administered to the subject, e.g. a human or animalpatient.

The active ingredients may be administered from 1 to 6 times a day,sufficient to exhibit the desired activity. These daily doses can begiven as a single dose once daily, or can be given as two or moresmaller doses at the same or different times of the day which in totalgive the specified daily dose. Preferably, the active ingredient isadministered once or twice a day. For instance, one dose could be takenin the morning and one later in the day.

In all aspects of the invention, the daily maintenance dose can be givenfor a period clinically desirable in the patient, for example from 1 dayup to several years (e.g. for the mammal's entire remaining life); forexample from about (2 or 3 or 5 days, 1 or 2 weeks, or 1 month) upwardsand/or for example up to about (5 years, 1 year, 6 months, 1 month, 1week, or 3 or 5 days). Administration of the daily maintenance dose forabout 3 to about 5 days or for about 1 week to about 1 year is typical.Other constituents of the liquid formulations may include preservatives,inorganic salts, acids, bases, buffers, nutrients, vitamins, or otherpharmaceuticals.

The human or animal subjects as taught herein may refer to human oranimal in need of therapy or treatment, comprising administering to thesaid human or animal a therapeutically effective amount of gram positivebacterium, preferably a lactic acid bacterium (LAB) or Bifidobacterium,as taught herein. The animal may preferably be a warm-blooded animal,more preferably a vertebrate, even more preferably a mammal, such as,e.g., domestic animals, farm animals, zoo animals, sport animals, petand experimental animals such as dogs, cats, guinea pigs, rabbits, rats,mice, horses, cattle, cows; primates such as apes, monkeys, orang-utans,and chimpanzees; canids such as dogs and wolves; felids such as cats,lions, and tigers; equids such as horses, donkeys, and zebras; foodanimals such as cows, pigs, and sheep; ungulates such as deer andgiraffes; rodents such as mice, rats, hamsters and guinea pigs; and soon. Generally, the term “subject” or “patient” may be usedinterchangeably and particularly refer to animals, preferablywarm-blooded animals, more preferably vertebrates, even more preferablymammals, still more preferably primates, and specifically includes humanpatients and non-human animals, mammals and primates. Preferred patientsmay be human subjects.

Further non-limiting examples of the types of diseases treatable inhumans or animals by delivery of gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, as described herein,optionally expressing prophylactic and/or therapeutic peptides,polypeptides or proteins, include, but are not limited to, e.g.,inflammatory bowel diseases including Crohn's disease and ulcerativecolitis (treatable with, e.g., IL-Ira or IL-10 or IL-27 or trefoilpeptides); autoimmune diseases, including but not limited to type-1diabetes, psoriasis, rheumatoid arthritis, lupus erythematosus(treatable with, e.g., IL-Ira or IL-10 or IL-27 or the relevantauto-antigen); allergic diseases including but not limited to asthma,food allergies, (treatable with the relevant allergen); celiac disease(treatable with gluten allergens and/or IL-27); neurological disordersincluding, but not limited to Alzheimer's disease, Parkinson's diseaseand amyotrophic lateral sclerosis (treatable with, e.g., brain deviatedneurotropic factor and ciliary neurotropic factor); cancer (treatablewith, e.g., IL-1, colony stimulating factors or interferon-W);osteoporosis (treatable with, e.g., transforming growth factor f3);diabetes (treatable with, e.g., insulin); cardiovascular disease(treatable with, e.g., tissue plasminogen activator); atherosclerosis(treatable with, e.g., cytokines and cytokine antagonists); hemophilia(treatable with, e.g., clotting factors); degenerative liver disease(treatable with, e.g., hepatocyte growth factor or interferon a);pulmonary diseases such as cystic fibrosis (treatable with, e.g., alphaantitrypsin); obesity; pathogen infections, e.g., viral or bacterialinfections (treatable with any number of the above-mentionedcompositions or antigens); etc.

The gram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, according to the invention can also be used to treatinfectious diseases. In an embodiment, passive immunization againstClostridium associated disease, preferably Clostridium difficileassociated disease (CDAD), with toxin-neutralizing antibodies locallyproduced and secreted via the gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, according to theinvention can be obtained. CDAD is mediated by two exotoxins, toxin A(enterotoxin; see for instance Genbank NC_009089.1, region:795843..803975 for DNA sequence or YP_001087137.1 for protein sequence)and toxin B (cytotoxin; see for instance Genbank NC_009089.1, region:787393..794493 for DNA sequence or YP_001087135.1 for protein sequence).Both are high-molecular-mass proteins that bind to the surface ofintestinal epithelial cells, where they are internalized and catalyzethe glucosylation of cytoplasmic rho proteins, leading to cell death,inflammation and diarrhea. They have also been implicated in promotingC. difficile virulence, colonization, and neutrophil chemotaxis andactivation. The bacteria itself is not invasive and does not causetissue damage. By neutralizing the C. difficile toxins with antibodies,the pathogenic mechanism of the pathogen is blocked, its ability tothrive in the gut may be diminished, and the impact on the microbialecology could be minimized, allowing recovery of the normal microflora.The medical advantage of this approach could include more rapidrecovery, fewer relapses, and relief from selective pressure forantibiotic resistance in normal gut flora. Accordingly, in a preferredembodiment, the gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, as described herein further contain,express, produce, and/or secrete neutralizing antibodies againstClostridium, preferably Clostridium dificile, toxin A and/or toxin B,wherein each of these toxins preferably has the sequence as indicatedabove. The skilled reader will understand that besides full lengthantibodies, various functional fragments or modified or variantantibodies may be used, as described herein elsewhere.

The skilled reader shall appreciate that the herein specifically reciteddiseases are only exemplary and their recitation is in no way intendedto confine the use of the gram positive bacterium, preferably a lacticacid bacterium (LAB) or Bifidobacterium, as taught herein, to theseparticular diseases. Instead, a skilled reader understands that the grampositive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, disclosed herein can be used to express in principleany expression products, preferably polypeptides, of interest, which maybe of therapeutic relevance in not only the recited ones but also invarious further diseases or conditions of humans and animals.Consequently, once a suitable expression product, preferably apolypeptide, e.g., an antigen and/or a prophylactically and/ortherapeutically gene product, polypeptide or protein, has been chosen ordetermined for a given ailment, a skilled person would be able toachieve its expression, isolation and/or delivery using the presentreagents.

The gram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, of the invention can be suspended in a pharmaceuticalformulation for administration to the human or animal having the diseaseto be treated. Such pharmaceutical formulations include but are notlimited to live host cells and a medium suitable for administration. Therecombinant host cells may be lyophilized in the presence of commonexcipients such as lactose, other sugars, alkaline and/or alkali earthstearate, carbonate and/or sulphate (for example, magnesium stearate,sodium carbonate and sodium sulphate), kaolin, silica, flavorants andaromas.

The gram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, according to the invention so-lyophilized may beprepared in the form of capsules, tablets, granulates and powders, eachof which may be administered by the oral route.

Alternatively, some gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, may be prepared as aqueoussuspensions in suitable media, or lyophilized bacteria may be suspendedin a suitable medium just prior to use, such medium including theexcipients referred to herein and other excipients such as glucose,glycine and sodium saccharinate.

For oral administration, gastroresistant oral dosage forms may beformulated, which dosage forms may also include compounds providingcontrolled release of the gram positive bacterium, preferably a lacticacid bacterium (LAB) or Bifidobacterium, and thereby provide controlledrelease of the desired protein encoded therein. For example, the oraldosage form (including tablets, pellets, granulates, powders) may becoated with a thin layer of excipient (usually polymers, cellulosicderivatives and/or lipophilic materials) that resists dissolution ordisruption in the stomach, but not in the intestine, thereby allowingtransit through the stomach in favour of disintegration, dissolution andabsorption in the intestine.

The oral dosage form may be designed to allow slow release of the grampositive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, (and optionally of the therapeutic and/orphrophylactice gene product thereof), for instance as controlledrelease, sustained release, prolonged release, sustained action tabletsor capsules. These dosage forms usually contain conventional and wellknown excipients, such as lipophilic, polymeric, cellulosic, insoluble,swellable excipients. Controlled release formulations may also be usedfor any other delivery sites including intestinal, colon, bioadhesion orsublingual delivery (i.e., dental mucosal delivery) and bronchialdelivery. When the compositions of the invention are to be administeredrectally or vaginally, pharmaceutical formulations may includesuppositories and creams. In this instance, the host cells are suspendedin a mixture of common excipients also including lipids. Each of theaforementioned formulations are well known in the art and are described,for example, in the following references: Hansel et al. (1990,Pharmaceutical dosage forms and drug delivery systems, 5th edition,William and Wilkins); Chien 1992, Novel drug delivery system, 2ndedition, M. Dekker); Prescott et al. (1989, Novel drug delivery, J.Wiley & Sons); Cazzaniga et al., (1994, Oral delayed release system forcolonic specific delivery, Int. J. Pharm. i08:7′).

Preferably, an enema formulation may be used for rectal administration.The term “enema” is used to cover liquid preparations intended forrectal use. The enema may be usually supplied in single-dose containersand contains one or more active substances dissolved or dispersed inwater, glycerol or macrogols or other suitable solvents.

A preferred embodiment of invention provides an enteric coated capsulecomprising stabilized freeze-dried, dried, or spray-dried viable grampositive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, as described herein characterized in that the viablegram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, are stabilized using a non-hygroscopic agent. As usedherein a non-hygroscopic agent is meant to include any excipienttypically used in the formulation of a pharmaceutical composition andwherein said agent exhibits an equilibrium moisture uptake at ambient40% RH of not more than about 8 wt %, preferably not more than about wt7%, and more preferably not more than about 6 wt %, for example about 1wt % to about 5 wt %, more in particular less or equal to 2 wt %. Thenon-hygroscopic agent can be a polyol such as for example mannitol,maltitol, isomalt (polyol sugar) or a phosphate salt such as for exampleanhydrous dicalcium phosphate dibasic calcium phosphate, calciumhydrogen phosphate, or for example a sugar such as sucrose.

The capsule used in the aforementioned formulation is typically selectedfrom the group consisting of a gelatin capsule, a starch capsule, ahydroxypropylmethylcellulose (HPMC) capsule and the like; in particulara HPMC capsule. For the intestinal delivery of viable bacteria, theenteric-coated capsules of the present invention should be stable at lowpH (up to pH 5.5) and have an accelerated dissolution profile at higherpH (above pH 5.5). The optimal release is realized when the capsulesdisintegrate at a pH of about 6.8 within 1 hour. Thus, in a furtherembodiment of the present invention the capsules are coated with anenteric polymer to provide an enteric coated capsule that is stable at apH up to 5.5 and that is soluble at a pH above 5.5; in particular at apH above 6.0; more in particular with a fast dissolution profile at a pHof about 6.8.

The enteric polymer used for the enteric coating typically consists of afilm-formable polymeric substance, which is soluble at a pH above 5.5,in particular at a pH above 6.0. Film-formable polymers useful in thedifferent embodiments of the present invention are usually selected fromthe group consisting of a cellulose derivative, an acrylic copolymer, amaleic copolymer, a polyvinyl derivative, shellac and the like; inparticular an acrylic copolymer selected from the group consisting ofstyrene-acrylic acid copolymer, methyl acrylate-acrylic acid copolymer,methyl acrylate-methacrylic acid copolymer, butylacrylate-styrene-acrylic acid copolymer, methacrylic acid-methylmethacrylate copolymer such as Eudragit L100, Eudragit S or EudragitS100 (each being trade name, commercially available from Röhm Pharma,Germany), methacrylic acid-ethyl acrylate copolymer such as EudragitL100-55 (trade name, commercially available from Röhm Pharma, Germany),methyl acrylate-methacrylic acid-octyl acrylate copolymer, and the like;more in particular the film-formable polymer consists of methacrylicacid-methyl methacrylate copolymer.

Also a combination of different stabilizing compounds (cryoprotectants)is added to the bacterial biomass before drying, spray-drying, orfreeze-drying. This combination of stabilizing compounds, comprising astarch hydrolysate and a glutamic acid salt and/or a polyol, results inimproved survival and stability of dried, spray-dried, or freeze-driedgram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium.

The formulations and capsules as described herein can be used as amedicament, nutraceutical, food additive, functional food, medical food,starter culture and/or probiotic composition.

Thus, according the invention, in a preferred embodiment, gram positivebacterium, preferably a lactic acid bacterium (LAB) or Bifidobacterium,as described herein, or the (pharmaceutical) compositions comprisingthese gram positive bacterium, preferably a lactic acid bacterium (LAB)or Bifidobacterium, may be administered to the animal or human viamucosal, e.g., an oral, nasal, rectal, vaginal or bronchial route by anyone of the state-of-the art formulations applicable to the specificroute. Dosages of gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, for administration will varydepending upon any number of factors including the type of bacteria andthe gene encoded thereby, the type and severity of the disease to betreated and the route of administration to be used. Thus, precisedosages cannot be defined for each and every embodiment of theinvention, but will be readily apparent to those skilled in the art oncearmed with the present invention. The dosage could be anyhow determinedon a case by case way by measuring the serum level concentrations of thetherapeutic and/or prophylactic protein after administration ofpredetermined numbers of cells, using well known methods, such as thoseknown as ELISA or Biacore (See examples). The analysis of the kineticprofile and half life of the delivered recombinant protein providessufficient information to allow the determination of an effective dosagerange for the gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium.

In an embodiment, when the gram positive bacterium, preferably a lacticacid bacterium (LAB) or Bifidobacterium, express an antigen, theinvention may thus also provide a vaccine. Preferably, the antigen maybe capable of eliciting an immune response in and used as a vaccine in ahuman or animal. The term “vaccine” identifies a pharmaceuticallyacceptable composition that, when administered in an effective amount toan animal or human subject is capable of inducing antibodies to animmunogen comprised in the vaccine and/or elicits protective immunity inthe subject. The vaccine of the invention would comprise the grampositive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, as taught herein, optionally transformed with thenucleic acids or vectors encoding the antigen and further optionally anexcipient. Such vaccines may also comprise an adjuvant, i.e., a compoundor composition that enhances the immune response to an antigen.Adjuvants include, but are not limited to, complete Freund's adjuvant,incomplete Freund's adjuvant, saponin, mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil or hydrocarbon emulsions, andpotentially useful pharmaceutically acceptable human adjuvants such asBCG (bacille Calmetle-Guerin) and Corynebacterium parvum.

In an aspect, the invention relates to a method for treatment or to atherapy, comprising administering the gram positive bacterium,preferably a lactic acid bacterium (LAB) or Bifidobacterium, orcompositions, preferably a therapeutic and/or prophylacticpharmaceutical composition to an individual in need thereof.

As described above, the composition comprising the gram positivebacterium, preferably a lactic acid bacterium (LAB) or Bifidobacterium,according to the invention may be a starter culture, a probioticcomposition, or a food additive. Accordingly, the invention in an aspectrelates to a starter culture, a probiotic composition, or a foodadditive comprising the gram positive bacterium, preferably a lacticacid bacterium (LAB) or Bifidobacterium, as described herein.

A starter culture may be, e.g., a liquid culture, liquid pressedculture, frozen or dried form, including, e.g., dried, freeze-dried formand spray/fluid bed dried form, or frozen or freeze-dried concentrated.Accordingly, in and embodiment, the invention relates to a starterculture as described herein, which is dried, spray-dried, frozen orfreeze-dried. The culture may be packed in vacuum, or under anatmosphere of, e.g., N₂, CO₂ and the like. For example, a starterculture may be produced and distributed in sealed enclosures, preferablynon-pyrogenic, which can be made of a rigid, non-flexible or flexiblesuitable plastic or other material, to the fermentation place and may beeither added to organic material to be fermented, or optionally firstcultivated in a separate starter medium to obtain a high densityculture.

A starter culture may also contain, in addition to the gram positivebacterium, preferably a lactic acid bacterium (LAB) or Bifidobacterium,according to the invention, buffering agents and growth stimulatingnutrients (e.g., an assimilable carbohydrate or a nitrogen source), orpreservatives (e.g., cryoprotective compounds) or other carriers, ifdesired, such as milk powder or sugars.

A starter culture may be a pure culture, i.e., may contain a biomass ofone single isolate of gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, according to the invention, i.e. aclone originating in principle from one cell. In another embodiment, astarter culture may be a co-culture, i.e., may comprise more than onestrain of gram positive bacterium, preferably a lactic acid bacterium(LAB) or Bifidobacterium, of the invention, optionally furthercomprising additional microorganisms such as bacteria or yeasts.

It may be preferred that a starter culture or a high density culturecontains at least 10² colony forming units (CFU) of one or more grampositive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, of the invention, such as at least 10³ CFU/g, at least10⁴ CFU/g, e.g., at least 10⁵ CFU/g, at least 10⁶ CFU/g, e.g., at least10⁷ CFU/g, at least 10⁸ CFU/g, e.g., at least 10⁹ CFU/g, at least 10¹⁰CFU/g, e.g., at least 10¹¹ CFU/g, at least 10¹² CFU/g, or at least 10¹³CFU/g.

Typically, a starter culture or a high density culture may be added to astarter medium or to organic material or substrate to be fermented in aconcentration of viable cells of one or more bacterial strains (andoptionally of one or more yeast strains) which is at least 10² (CFU) ofone or more bacterial strains (and optionally of one or more yeaststrains) of the invention, such as at least 10³ CFU/g, at least 10⁴CFU/g, e.g., at least 10⁵ CFU/g, at least 10⁶ CFU/g, e.g., at least 10⁷CFU/g, at least 10⁸ CFU/g, e.g., at least 10⁹ CFU/g, at least 10¹⁰CFU/g, e.g., at least 10¹¹ CFU/g, at least 10¹² CFU/g, or at least 10¹³CFU/g of the organic material, medium or substrate.

In an embodiment, the invention relates to a starter culture as definedherein for the preparation of a food product or relates to a foodadditive or a probiotic composition, comprising a gram positivebacterium, preferably a lactic acid bacterium (LAB) or Bifidobacterium,lacking trehalose 6-phosphate phosphorylase (TrePP) activity. In apreferred embodiment the gene encoding endogenous TrePP has beenpartially or completely deleted, disrupted or inactivated such as beingincapable of producing functional trePP gene product, as describedherein elsewhere. In a further embodiment, the gram positive bacterium,preferably a lactic acid bacterium (LAB) or Bifidobacterium, in thestarter culture, the probiotic composition, or the food additive doesnot contain functional heterologous trehalose 6-phosphate phosphatase(e.g. otsB) and/or a functional heterologous trehalose 6-phosphatesynthase (e.g. otsA), as described herein elsewhere. In yet a furtherembodiment, the gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, in the starter culture, probioticcomposition, or food additive as described herein does not contain afunctional heterologous gene involved in the metabolism (eithercatabolic or anabolic) of trehalose or trehalose 6-phosphate. Such genesencompass, without limitation trehalase, trehalose phosphorylase, andtrehalose 6-phosphate hydrolase.

In another embodiment, the invention relates to a starter culture,probiotic composition, or food additive as defined herein, wherein thegram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, lacks cellobiose-specific PTS system IIC component(ptcC) activity. In a preferred embodiment the gene encoding endogenousthe gene encoding endogenous ptcC has been partially or completelydeleted, disrupted or inactivated such as being incapable of producingfunctional ptcC gene product, as described herein elsewhere.

In a further embodiment, the invention relates to a starter culture,probiotic composition, or food additive as described herein, wherein thegram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, overexpresses, preferably constitutively overexpresses,one or more genes encoding a trehalose transporter, preferably anendogenous trehalose transporter, as described herein elsewhere.

In yet another embodiment, the invention relates to a starter culture,probiotic composition, or food additive as described herein, wherein thegram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, expresses one or more heterologous gene product,preferably one or more prophylactic and/or therapeutic gene product, asdescribed herein elsewhere.

In an embodiment, the starter culture, probiotic composition, or foodadditive as described herein is dried, frozen, spray-dried orfreeze-dried.

As indicated above, in an aspect, the invention relates to the use ofthe gram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, as described herein for the preparation of a starterculture, preferably for use as a food additive or probiotic compositionor for use in the preparation of a food product. In a further aspect,the invention relates to the use of a starter culture as a foodadditive, probiotic composition or for the preparation of a foodproduct.

As used herein, the term “food additive” refers to gram positivebacterium, preferably a lactic acid bacterium (LAB) or Bifidobacterium,preferably formulated in a composition, which can be added to a human oranimal food or feed, suitable for consumption without furthermodification or alternatively after further modification, such ascomplete or partial fermentation of the food or feed or the complete orpartial fermentation of one or more components of the food or feed. Bymeans of example, and without limitation, the gram positive bacterium,preferably a lactic acid bacterium (LAB) or Bifidobacterium, accordingto the invention, or the compositions according to the invention, suchas the starter cultures described herein, may be used in the dairyindustry, in particular for the preparation of fermented milk products,also known as cultured dairy foods, cultured dairy products, or culturedmilk products. The fermentation process increases the shelf-life of theproduct, as well as adds to the taste and improves the digestibility ofmilk. Examples of food products referred to herein, include, but are notlimited to cheese, yoghurt, sour cream, buttermilk, acidophilus milk, .. . .

In an aspect, the invention also relates to a method for preparing amedicament, a food additive, a probiotic composition, or a starterculture as defined herein, wherein said starter culture is preferably astarter culture for the preparation of a food product, comprising thesteps of:

-   i) propagating gram positive bacterium, preferably a lactic acid    bacterium (LAB) or Bifidobacterium, as defined herein in a medium    comprising a substrate material capable of being fermented by said    gram positive bacterium, preferably a lactic acid bacterium (LAB) or    Bifidobacterium; and-   ii) formulating the so propagated gram positive bacterium,    preferably a lactic acid bacterium (LAB) or Bifidobacterium, as a    medicament, food additive, probiotic composition, or starter    culture, respectively.

Methods for propagating gram positive bacterium, preferably a lacticacid bacterium (LAB) or Bifidobacterium, as well as media and substratescapable of being fermented by gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, are well known in theart. In an embodiment, the formulation as a medicament, food additive,probiotic composition, or starter culture comprises formulating as aliquid culture, liquid pressed culture, frozen or dried form, including,e.g., dried, freeze-dried form and spray/fluid bed dried form, or frozenor freeze-dried concentrated. Preferably, the formulation comprisesdrying, spray-drying, freezing or freeze-drying.

In a further aspect, the invention also relates to a method forpreparing a food product, comprising the step of admixing the grampositive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, as defined herein, the food additive as defined herein,the probiotic composition as defined herein, or the starter culture asdefined herein with a substrate material capable of being fermented bysaid gram positive bacterium, preferably a lactic acid bacterium (LAB)or Bifidobacterium. The substrate material is typically a carbon source,preferably a carbohydrate or sugar. Carbohydrates capable of beingfermented by gram positive bacterium, preferably a lactic acid bacterium(LAB) or Bifidobacterium, include, but are not limited tomonosaccharides or disaccharides such as glucose, fructose, galactose,sucrose, lactose, maltose, trehalose, cellobiose, . . . . In anembodiment, the method for preparing a food product comprises the stepsof:

-   i) providing the gram positive bacterium, preferably a lactic acid    bacterium (LAB) or Bifidobacterium, food additive, probiotic    composition, or the starter culture as described herein;-   ii) providing a substrate material or a composition, preferably a    non-toxic or an edible composition, comprising a substrate material    which is capable of being fermented by said gram positive bacterium,    preferably a lactic acid bacterium (LAB) or Bifidobacterium;-   iii) admixing the gram positive bacterium, preferably a lactic acid    bacterium (LAB) or Bifidobacterium, as defined herein, food additive    as defined herein, the probiotic composition as defined herein, or    the starter culture as defined herein with the substrate material or    composition-   iv) optionally propagating said gram positive bacterium, preferably    a lactic acid bacterium (LAB) or Bifidobacterium, and/or fermenting    said substrate material or composition with said gram positive    bacterium, preferably a lactic acid bacterium (LAB) or    Bifidobacterium.

In an aspect, the invention also relates to a food product directly orindirectly obtained or obtainable by the herein described methods.

As described before, the gram positive bacterium, preferably a lacticacid bacterium (LAB) or Bifidobacterium, according to the inventionadvantageously accumulate trehalose intracellularly. Accordingly, in anaspect, the invention also relates to a method for internallyaccumulating trehalose in a gram positive bacterium, preferably a lacticacid bacterium (LAB) or Bifidobacterium, such as trehalose being presentin the growth medium, or externally or exogenously added trehalose,comprising the step of propagating gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, according to theinvention in a medium comprising a substrate material capable of beingfermented by said gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium. In an embodiment, the method forinternally accumulating trehalose in a gram positive bacterium,preferably a lactic acid bacterium (LAB) or Bifidobacterium, comprisesthe steps of:

-   i) providing the gram positive bacterium, preferably a lactic acid    bacterium (LAB) or Bifidobacterium, or the starter culture as    described herein;-   ii) providing a substrate material or a composition, preferably a    non-toxic or an edible composition, comprising a substrate material    which is capable of being fermented by said gram positive bacterium,    preferably a lactic acid bacterium (LAB) or Bifidobacterium;-   iii) admixing the gram positive bacterium, preferably a lactic acid    bacterium (LAB) or Bifidobacterium, as defined herein, or the    starter culture as defined herein with the substrate material or    composition-   iv) optionally propagating said gram positive bacterium, preferably    a lactic acid bacterium (LAB) or Bifidobacterium, and/or fermenting    said substrate material or composition with said gram positive    bacterium, preferably a lactic acid bacterium (LAB) or    Bifidobacterium.

The gram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, according to the invention advantageously show animproved resistance to stress as well as improved manufacturing,processing and/or storage characteristics. Accordingly, in an aspect,the invention relates to a method for improving stress resistance ormanufacturing, processing and/or storage characteristics of a grampositive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, comprising modifying the gram positive bacterium,preferably a lactic acid bacterium (LAB) or Bifidobacterium, such as tolack TrePP activity. In an embodiment, the gene encoding endogenousTrePP has been partially or completely deleted, disrupted or inactivatedsuch as being incapable of producing functional trePP gene product.Preferably the stress resistance or manufacturing, processing and/orstorage characteristics is one or more stress resistance ormanufacturing, processing and/or storage characteristics selected fromthe group comprising resistance to acid conditions, resistance to bilesalts, resistance to heat, resistance to salt, resistance to drying,spray-drying, freezing or freeze-drying, and osmotic resistance.

In an embodiment, the invention relates to any of the methods asdescribed herein, wherein the gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, does not containfunctional heterologous trehalose 6-phosphate phosphatase (e.g. otsB)and/or a functional heterologous trehalose 6-phosphate synthase (e.g.otsA), as described herein elsewhere. In yet a further embodiment, theinvention relates to any of the methods as described herein, wherein thegram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, does not contain a functional heterologous geneinvolved in the metabolism (either catabolic or anabolic) of trehaloseor trehalose 6-phosphate. Such genes encompass, without limitationtrehalase, trehalose phosphorylase, and trehalose 6-phosphate hydrolase.

In another embodiment, the invention relates any of the methods asdescribed herein, wherein the gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, lackscellobiose-specific PTS system IIC component (ptcC) activity. In apreferred embodiment the gene encoding endogenous the gene encodingendogenous ptcC has been partially or completely deleted, disrupted orinactivated such as being incapable of producing functional ptcC geneproduct, as described herein elsewhere.

In a further embodiment, the invention relates to any of the methods asdescribed herein, wherein the gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, overexpresses,preferably constitutively overexpresses, one or more genes encoding atrehalose transporter, preferably an endogenous trehalose transporter,as described herein elsewhere.

In yet another embodiment, the invention relates to any of the methodsas described herein, wherein the gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, expresses one or moreheterologous gene product, preferably one or more prophylactic and/ortherapeutic gene product, as described herein elsewhere.

In an embodiment, the invention relates to any of the methods asdescribed herein, further comprising the step of drying, freezing,spray-drying, or freeze-drying the gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, medicament, foodadditive, probiotic composition, or starter culture.

The inventors have surprisingly found that the gram positive bacterium,preferably a lactic acid bacterium (LAB) or Bifidobacterium, accordingto the invention are capable of intracellularly accumulating trehalosewithout the addition of externally added trehalose. Advantageously, thegram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, according to the invention can be propagated in mediumoptionally even without externally added trehalose but still accumulatetrehalose internally. Accordingly, in an embodiment, the inventionrelates to the methods as described herein, comprising the step ofmaintaining or propagating the gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, as described herein in amedium lacking or substantially lacking trehalose, or a medium lackingor substantially lacking externally or exogenously added trehalose.Advantageously, such medium can comprise another fermentable carbonsource, such as, but without limitation maltose and/or glucose.

Accordingly, in an embodiment, the invention relates to any of themethods as described herein, wherein the gram positive bacterium,preferably a lactic acid bacterium (LAB) or Bifidobacterium, accordingto the invention are maintained or propagated in a medium comprising asubstrate material capable of being fermented by said gram positivebacterium, preferably a lactic acid bacterium (LAB) or Bifidobacterium,wherein said substrate material comprises less (such as suboptimal),does not comprise or substantially does not comprise trehalose.Alternatively, the invention relates to any of the methods as describedherein, wherein the gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, according to the invention aremaintained or propagated in a medium comprising a substrate materialcapable of being fermented by said gram positive bacterium, preferably alactic acid bacterium (LAB) or Bifidobacterium, wherein said substratematerial comprises maltose. In a preferred embodiment, the inventionrelates to any of the methods as described herein, wherein the grampositive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, according to the invention are maintained or propagatedin a medium comprising a substrate material capable of being fermentedby said gram positive bacterium, preferably a lactic acid bacterium(LAB) or Bifidobacterium, wherein said substrate material comprisesmaltose and wherein said substrate material comprises less (such assuboptimal), does not comprise or substantially does not comprisetrehalose.

As used herein, a medium comprising no or substantially no trehalose orno externally added or exogenous trehalose refers to a medium which doesnot contain trehalose or which only contains small quantities oftrehalose. Preferably, the amount or concentration of trehalose in suchmedium is too low to allow for the bacteria to be able to use as a solecarbon source. In an embodiment, the medium contains less than 100 mM,preferably less than 50 mM, more preferably less than 25 mM, such asless than 15 mM, less than 10 mM, less than 5 mM, less than 2 mM, orless than 1 mM. In a further embodiment, the medium contains less than 2w/w % or less than 2 v/w % trehalose, preferably less than 1 w/w % orless than 1 v/w % trehalose, more preferably less than 0.5 w/w % or lessthan 0.5 v/w % trehalose, such as less than 0.3, less than 0.2, lessthan 0.1, less than 0.05, or less than 0.01 w/w % or v/w % trehalose. Inanother embodiment, the medium contains less than 20% trehalose of thetotal amount of carbon source or fermentable carbohydrate, preferablyless than 10%, more preferably less than 5%, such as less than 3%, lessthan 2%, or less than 1%.

In a further aspect, the invention relates to the use of the grampositive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, as described herein to accumulate intracellulartrehalose in said gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium. In a preferred embodiment, theinvention relates to the use of the gram positive bacterium, preferablya lactic acid bacterium (LAB) or Bifidobacterium, as described herein toaccumulate intracellular trehalose in said gram positive bacterium,preferably a lactic acid bacterium (LAB) or Bifidobacterium, in theabsence or substantial absence of trehalose. In another embodiment, theinvention relates to the use of the gram positive bacterium, preferablya lactic acid bacterium (LAB) or Bifidobacterium, as described herein toaccumulate intracellular trehalose in said gram positive bacterium,preferably a lactic acid bacterium (LAB) or Bifidobacterium, whenmaintained or propagated on maltose, preferably as the sole orsubstantially sole carbon source.

As described above, the gram positive bacterium, preferably a lacticacid bacterium (LAB) or Bifidobacterium, according to the invention showan improved resistance to a variety of environmental stresses as well asimproved manufacturing, processing and/or storage characteristics.Accordingly, in an aspect, the invention relates to the use of the grampositive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, as described herein to improve stress resistance or toimprove manufacturing, processing and/or storage characteristics in saidgram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium. In a further aspect, the invention relates to a methodfor improving stress resistance or for improving manufacturing,processing and/or storage characteristics in gram positive bacterium,preferably a lactic acid bacterium (LAB) or Bifidobacterium, comprisinggenerating the gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, as described herein.

In an embodiment, the stress resistance is selected from the groupcomprising resistance to acid conditions, resistance to bile salts, heatresistance, resistance to salt, cold resistance, osmotic resistance,preferably selected from resistance to acid conditions or bile salts,more preferably resistance to bile salts. In another embodiment, themanufacturing, processing and/or storage characteristics are selectedfrom the group comprising drying, freezing, freeze-drying, spray-dryingor storage in medium, preferably freezing or freeze-drying, morepreferably freeze-drying.

In another aspect, the invention relates to a gram positive bacterium,preferably a lactic acid bacterium (LAB) or Bifidobacterium, lackingTrePP activity and further displaying any one or preferably both of thefollowing characteristics: (i) the gram positive bacterium lacks ptcCactivity; (ii) the gram positive bacterium overexpresses, preferablyconstitutively overexpresses, one or more trehalose transporters. In anembodiment, the invention relates to a gram positive bacterium,preferably a lactic acid bacterium (LAB) or Bifidobacterium, lackingTrePP activity and lacking ptcC activity. In another embodiment, theinvention relates to a gram positive bacterium, preferably a lactic acidbacterium (LAB) or Bifidobacterium, lacking TrePP activity and(constitutively) overexpressing one or more trehalose transporters. Inyet another embodiment, the invention relates to a gram positivebacterium, preferably a lactic acid bacterium (LAB) or Bifidobacterium,lacking TrePP activity, lacking ptcC activity, and (constitutively)overexpressing one or more trehalose transporters. In a furtherembodiment, the invention relates to any of these gram positivebacterium, preferably a lactic acid bacterium (LAB) or Bifidobacterium,which do not contain functional heterologous trehalose 6-phosphatephosphatase and/or functional heterologous trehalose 6-phosphatesynthase. In another embodiment, the invention relates to any of thesegram positive bacterium, preferably a lactic acid bacterium (LAB) orBifidobacterium, which contain functional heterologous trehalose6-phosphate phosphatase and/or functional heterologous trehalose6-phosphate synthase. In a further embodiment, the invention relates toany of these gram positive bacterium, preferably a lactic acid bacterium(LAB) or Bifidobacterium, containing one or more heterologous geneproducts, preferably prophylactic and/or therapeutic gene product(s),preferably not containing functional heterologous trehalose 6-phosphatephosphatase and/or functional heterologous trehalose 6-synthase.

The aspects and embodiments of the invention are further supported bythe following non-limiting examples.

EXAMPLES

Table 1 provides an overview of genetic modifications in strainsdescribed herein, except for strains used in FIG. 4 which are given inTable 2.

TABLE 1 a) trehalose operon trehalose strain PTS I/II TrePP b) ptcC c)otsB d) thyA e) Cargo sAGX0037 wt wt wt — KO (gene PhllA>>hIL-10(Ptre>>PTS) replacement) (thyA locus) sAGX0085 wt wt wt — KO (genePhllA>>hTFF1 (Ptre>>PTS) replacement) (thyA locus) sAGX0137 wt wt wtusp45>> KO (gene PhllA>>hIL-10 (Ptre>>PTS) mutant otsB replacement)(thyA locus) sAGX0139 wt wt wt usp45>>otsB KO (gene PhllA>>hIL-10(Ptre>>PTS) replacement) (thyA locus) sAGX0147 wt KO wt usp45>> KO (genePhllA>>hIL-10 (Ptre>>PTS) mutant otsB replacement) (thyA locus) sAGX0148wt KO wt usp45>>otsB KO (gene PhllA>>hIL-10 (Ptre>>PTS) replacement)(thyA locus) sAGX0167 PhllA>>PTS KO wt usp45>>otsB KO (genePhllA>>hIL-10 replacement) (thyA locus) sAGX0169 wt KO wt — KO (genePhllA>>hTFF-1 (Ptre>>PTS) replacement) (thyA locus) sAGX0248 wt KO KO(stop — wt — (Ptre>>PTS) codon) sAGX0272 wt KO wt — wt — (Ptre>>PTS)sAGX0309 PhllA>>PTS KO wt — KO (gene usp45>>CDP870 deletion) anti-TNFsAGX0319 KO KO wt — KO (gene usp45>>CDP870 deletion) anti-TNF sAGX0346PhllA>>PTS KO KO (stop usp45>>otsB KO (gene — codon) deletion) sAGX0347PhllA>>PTS KO KO (gene usp45>>otsB KO (gene — deletion) deletion)sAGX0354 PhllA>>PTS KO KO (stop usp45>>otsB KO (gene gapB>>CDP870 codon)deletion) anti-TNFOverview of genetic modifications in strains described herein. Indicatedis a) the structure of the trehalose operon: whether the nativetrehalose operon promoter (Ptre) precedes the trehalose PTS transporters(Ptre>>PTS) and whether trePP was deleted (KO) or not (wild type; wt);b) structure of the ptcC gene: wild type (wt), inactivated (KO) byinsertion of a stop codon or gene deletion; c) absence (−) or presenceof functionally inactive (mutant) otsB or wild type otsB, eitherinserted at the thyA locus following the thyA promoter (PthyA>>otsB) orinserted as a second cistron following the usp45 gene (usp45>>otsB); d)structure of the thyA gene: wild type (wt) or inactivated (KO) by genedeletion or insertion of a cargo gene (gene replacement); e) absence (−)or nature and structure of uidA, hIL-10 or anti-TNF CDP870 cargo genes,inserted at the thyA locus under control of the hIIA promoter (PhIIA>>)or inserted downstream of the usp45 or gapB genes (usp45>>; gapB>>). Allstrains are derived from L. lactis MG1363.

TABLE 2 Inactivated Inactivated Strain Gene Gene ID Inactivated proteinproduct sAGX0241 pmrB 4799106 multidrug resistance efflux pump sAGX0242celB 4796591 cellobiose-specific PTS system IIC component sAGX0245 araJ4796972 putative arabinose efflux permease sAGX0246 ptcB 4797109cellobiose-specific PTS system IIB component sAGX0247 ptcA 4798642cellobiose-specific PTS system IIA component sAGX0248 ptcC 4796893cellobiose-specific PTS system IIC component sAGX0249 msmK 4797024multiple sugar-binding transport ATP-binding protein sAGX0250 llmg_04534797778 sucrose-specific PTS enzyme IIABC (tre operon) sAGX0251llmg_0454 4797093 beta-glucoside-specific PTS system IIABC component(tre operon) sAGX0252 llmg_0489 4796717 sugar transport system permeaseprotein sAGX0253 llmg_0490 4796719 sugar transport system permeaseprotein sAGX0255 malG 4798664 maltose ABC transporter permease proteinmalG sAGX0256 malF 4798442 maltose transport system permease proteinmalF sAGX0257 malE 4798313 maltose ABC transporter substrate bindingprotein sAGX0258 lplB 4798767 sugar ABC transporter substrate bindingprotein sAGX0259 lplC 4796680 sugar ABC transporter permease sAGX0260lplA 4797636 sugar ABC transporter substrate-binding protein sAGX0261bglP 4797495 PTS system, beta-glucosides specific enzyme IIABC sAGX0262llmg_1104 4798113 drug-export protein sAGX0265 tagG 4798685 teichoicacid ABC transporter permease proteinOverview of strains constructed to identify the trehalose exit port.Strains were constructed that are deficient in trePP to allow trehaloseaccumulation and in which a selection of genes, taken from L. lactisCOGs functional categories “Carbohydrate transport and metabolism”http://www.ncbi.nlm.nih.gov/sutils/cogtik.cgi?gi=20494&cog=G (from whichgene and protein nomenclature was taken) are inactivated. The Gene ID ofinactivated genes is indicated as well as the inactivated proteinproduct.

Gene inactivation was performed by oligonucleotide directedrecombineering, introducing in-frame stopcodons in the respective targetgenes. All strains are derived from L. lactis MG1363

Example 1: Intracellular Trehalose Accumulation Following trePPInactivation

Experimental

Strains were grown overnight (A) or for 24 hours (B) in 50 ml GM17T+500mM trehalose at 30° C., cells were collected by centrifugation andtrehalose content was determined: equivalents of 10 ml overnight culturewere washed 3 times with 0.25 M carbonate buffer where after weight ofthe cell pellet (wet weight) was determined. Cells were lysed in 1 ml0.25 M carbonate buffer using the lysing matrix B and the MP Fasprep-24device at 6 m/s for 40 seconds (MP Biomedicals). Supernatant of thelysed cells was separated by centrifugation and heated for 30 minutes at99° C. Cell debris was removed by centrifugation and the supernatant wasassayed for trehalose concentration using a trehalose assay kit (K-TREH010/10, Megazyme, Ireland). Briefly, trehalose in the samples ishydrolysed to D-glucose by trehalase, and the D-glucose released isphosphorylated by the enzyme hexokinase and adenosine-5′triphosphate(ATP) to glucose-6-phosphate with the simultaneous formation ofadenosine-5′diphosphate (ADP). In the presence of the enzymeglucose-6-phosphate dehydrogenase, glucose-6-phosphate is oxidized bynicotinamide-adenine dinucleotide phosphate (NADP+) togluconate-6-phosphate with the formation of reduced nicotinamide-adeninedinucleotide phosphate (NADPH). The amount of NADPH formed in thisreaction is stoichiometric with the amount of D-glucose and thus withthe amount of trehalose. It is the NADPH which is measured by theincrease in absorbance at 340 nm (in comparison to the OD340 before theaddition of trehalose). Trehalose values were calculated by use of aserial dilution of a trehalose standard and expressed as mg/g wet cellpellet weight (ww)

Results

Intracellular trehalose accumulation is possible following trePPinactivation, following otsB expression or a combination thereof, asindicated in FIG. 1. FIG. 1 (A) depicts trePP wild type strains(sAGX0037 and sAGX0137) do not accumulate trehalose. Inactivation oftrePP in sAGX0137 (containing a non-functional mutant otsB), leading tosAGX0147, allows for the accumulation of trehalose. Insertion of wildtype otsB sAGX0037, leading to sAGX0139, allows for the accumulation oftrehalose. Combination of otsB and trePP KO leads to a moderate increasein trehalose accumulation (sAGX0148) which is greatly potentiated by theinsertion of the strong constitutive PhIIA promoter (which is disclosedin WO 2008/084115) in front of both phosphotransferase system (PTS)genes of the L. lactis trehalose operon (sAGX0167). FIG. 1 (B) showsthat trePP wild type strain sAGX0085 cannot accumulate trehalose.Inactivation of trePP KO (sAGX0169) only allows for the accumulation oftrehalose.

From FIG. 1 it is clear that trePP wild type strains do not accumulatetrehalose. Gene disruption (gene deletion but also point mutation) oftrePP allows intracellular accumulation of exogenous trehalose. Instrain sAGX0147 a non-functional otsB mutant gene is present, whilestrains sAGX0169, sAGX0309 and sAGX0319 carry no otsB genes. StrainsAGX0169 carries, except for the hTFF1 cargo gene present in the thyAlocus, no other genetic alteration than the disruption of trePP.Trehalose accumulation in a trePP KO strain is unexpected as one woulddeem, according to the prior art, this to be critically dependent on atrehalose-6-phosphate phosphatase (otsB or analogue). Such function hasnot been described in L. lactis and would not be expected to be presentas it would counteract the metabolism of trehalose by L. lactis byconverting trehalose-6-phosphate to the inert intracellular trehalose.We here observe that, unexpectedly, this function is present in L.lactis. trePP KO can be performed by gene deletion, as was done here orby the establishment of a stop codon or frame shift mutation or apromoter mutation or the identification of a spontaneous non-functionaltrePP mutant. Trehalose accumulation is possible when otsB is present assuch (sAGX0139) or combined with trePP KO (sAGX0148) or even furthercombined with an insertion of the strong constitutive promoter PhIIApositioned in front of both phosphotransferase system (PTS) genes(PhIIA>>trePTC) of the L. lactis trehalose operon (sAGX0167). Thepreferred position of otsB, as it is used here, is as a second cistronbehind the indigenous usp45 gene in a configuration as describedEuropean patent applications with application numbers 11168495.7 and11173588.2 (usp45>>rpmD>>otsB, wherein rpmD is the intergenic regionpreceding rpmD).

Example 2: The Accumulation of Exogenous Trehalose Provides ProtectionTowards Bile Lysis

Experimental

Strains were grown overnight in 50 ml GM17T or GM17T+500 mM trehalose at30° C., cells were collected by centrifugation and resuspended in 25 ml0.9% NaCl. Samples were taken and CFU were determined by platingappropriate dilutions (initial) At T0, 25 ml 1% oxgal in 0.9% NaCl wasadded and cell suspensions were incubated for 8 h at 37° C. Samples weretaken at T0, 1, 2, 4, 6 and 8 h. CFU were determined by platingappropriate dilutions (FIG. 2 A), trehalose content was determined (FIG.2 B) essentially as described in Example 1

Results

Intracellular trehalose protects against bile lysis and the loss ofintracellular trehalose coincides with decreased resistance to bilelysis. Therefore, leakage of trehalose is problematic for long termstability in bile.

Indicated in FIG. 2 is that the accumulation of exogenous trehalose inL. lactis cells provides protection towards bile lysis. Release ofintracellular trehalose limits the protective effect of trehalose intime. L. lactis cells that have accumulated trehalose(sAGX0167+trehalose, sAGX0309+trehalose and sAGX0319+trehalose) i.e.grown in 500 mM trehalose as described in (FIG. 2 B) show a substantialprotection in time against bile lysis, proportional to the concentrationof intracellular trehalose when compared to L. lactis cells withoutintracellular trehalose (sAGX0167, precultured without trehalose).Decreasing survival in 0.5% oxgal (FIG. 2 A) coincides with release ofintracellular trehalose (FIG. 2 B).

Example 3: The Accumulation of Exogenous Trehalose in ProvidesProtection Towards Bile Lysis

Experimental

Cells were collected by centrifugation and resuspended in 1×M9 saltssolution. Samples were taken and trehalose concentrations weredetermined at T0, 1, 2 and 4 hours, essentially as described inExample 1. Data are exemplary for all ptcC wt strains.

Results

Following accumulation, Trehalose to some extent leaks from cellsthrough an up to now unidentified or unanticipated trehalose exit portand can be recovered in the supernatant.

FIG. 3 A indicates that trehalose can be accumulated intracellular byde-novo synthesis as well as following uptake from the growth medium(sAGX0167 grown in 500 mM trehalose, as described in FIG. 1). Bothde-novo synthesized as well as exogenously accumulated trehalose arereleased from the cells. FIG. 3 B indicates that loss of intracellulartrehalose results in increase of trehalose present in the culturesupernatant (here expressed as mg trehalose/10 ml culture to allowcomparison between intracellular and extracellular trehaloseconcentration).

Example 4: Trehalose Accumulation and Release in Various StrainsDescribed in Table 2

Experimental

Strains described in Table 2 were grown in GM17 supplemented with 100 mM(FIG. 4 A) or 500 mM (FIG. 4 B) trehalose. Cells were collected andresuspended in M9 buffer (Difco). Intracellular trehalose content wasdetermined at T0, 2, 4 and 8 h, essentially as described in Example 1.Except for sAGX0248 (ptcC KO) all strain show a similar release oftrehalose as described in FIG. 3.

Results

20 L. lactis MG1363 oligosacharride transporters were selected from COGdatabase (section Carbohydrate transport and metabolism) and their geneswere disrupted by oligonucleotide directed recombineering in a trePP KObackground (sAGX0272; required for trehalose accumulation) (Table 2;FIG. 4). Only the disruption of ptcC circumvents the release oftrehalose.

One cannot predict which of the genes listed in Table 2 is involved intrehalose release. Disruption of either one of the PTS transporter genespresent in the trehalose operon (IImg_0453; IImg_0454) has no effect ontrehalose uptake or release. Disruption of the ptcC gene (encodingcellobiose-specific PTS system IIC component) resolves leakage ofaccumulated trehalose, therefore the PtcC is the trehalose exit port andthis protein causes leakage of trehalose. Disruption of celB(cellobiose-specific PTS system IIC component) has no effect ontrehalose uptake or release. Disruption of ptcC in trePP KO backgroundprevents all release of trehalose.

Example 5: Trehalose Accumulation and Release in Various StrainsDescribed in Table 2

Experimental

Strains were grown overnight in GM17T+500 mM trehalose at 30° C., cellswere collected by centrifugation and resuspended in an equal volume 1×M9(FIG. 5 A) or 0.5% Oxgal in 0.9% NaCl (FIG. 5 B) and incubated for 24 hat 37° C. Samples were taken at T0, 1, 2, 4, 6, 8, 12 and 24 h.Intracellular trehalose content was determined as described in Example1.

Results

Combined ptcC KO (stop codon insertion as well as gene deletion) andPhIIA>>trePTC (constitutive high expression of trehalose transporter)allows for high trehalose import and full intracellular retention.

FIG. 5 indicates that inactivation of ptcC prevents (in M9 salts, panelA) or delays (in 0.5% oxgal, panel B) the release of intracellulartrehalose. Presence of the strong constitutive PhIIA promoter (asdisclosed in WO 2008/084115, which incorporated herein in its entiretyby reference) in front of both PTS genes of the L. lactis trehaloseoperon restores the capacity to accumulate exogenous trehalose to thatof a reference strain (see also FIG. 4).

Example 6: The Accumulation of Exogenous Trehalose Provides ProtectionTowards Bile Lysis

Experimental

Strains were grown overnight in GM17T+500 mM trehalose at 30° C., cellswere collected by centrifugation and resuspended in half a volume 0.9%NaCl. Samples were taken and CFU were determined by plating appropriatedilutions (initial). At T0, half a volume 1% oxgal in 0.9% NaCl wasadded and incubated for 8 h at 37° C. Samples were taken at T0, 1, 2, 4,6, 8, 12 and 24 hours. Trehalose content was determined (FIG. 6 A,essentially as in Example 1) and CFU were determined by plating (0-8hours only) appropriate dilutions and plotted as % of initial T0 values(FIG. 6 B).

Results

The enhanced capacity to retain intracellular trehalose leads toimproved bile resistance.

FIG. 6 indicates that the accumulation of exogenous trehalose in L.lactis cells provides protection towards bile lysis. Release ofintracellular trehalose (A) coincides with decreasing survival in 0.5%oxgal (B). Inactivation of ptcC extends the presence in time ofintracellular trehalose and consequently also improves resistance intime towards oxgal.

Example 7: trePP KO Strains are Capable of Converting Glucose or Maltoseto Intracellular Trehalose. Maltose Stimulates Trehalose Uptake by trePPKO Strains

Experimental

Strains were grown overnight in the indicated media. Trehalose wasdetermined essentially as described in Example 1.

Results

trePP KO strains have acquired the capacity to utilize carbon sourcessuch as glucose or maltose to accumulate trehalose. This is notdescribed in the prior art as trehalose can accumulate inside the cellsin MM17T i.e. with maltose as the single carbon source.

FIG. 7 indicates that trePP KO strains (both ptcC wt as well as ptcC KO)are capable of converting glucose or maltose to intracellular trehalose(columns 1 and 2, columns 5-8). Maltose enhances the uptake andaccumulation of extracellular trehalose from the growth medium in trePPKO strains (columns 9 and 10).

trePP KO strains accumulate trehalose when grown:

-   1. With glucose as a carbon source (GM17T; columns 1 and 2)-   2. With glucose as a carbon source and extracellular trehalose    (GM17T+500 mM trehalose; columns 3 and 4)-   3. With maltose as a carbon source (MM17T; columns 5 and 6)-   4. With glucose and maltose as a carbon source (GM M17T; columns 7    and 8)-   5. With maltose as a carbon source and extracellular trehalose    (MM17T+500 mM trehalose; columns 9 and 10)

Example 8: Survival after Lyophilization

Experimental

Table 3 A: Growth optimized 200 L culture (animal protein freefermentation medium) was 10 fold concentrated through ultrafiltrationand diafiltration and re-suspension in concentrated cryoprotectant mix(as described in WO 2010/124855). CFU count per ml was determined forthe bacterial suspension. The suspension was filled out in bulk andanalytical trays, trays were weighed and lyophilized. For viabilityassessment, lyophilized appropriate weight portions were reconstitutedwith appropriate volumes of purified water and CFU count per ml wasdetermined. Viability % was determined from the ratio of CFU before andafter lyophilization.

Table 3 B: Overnight 20 L culture (GM17T+500 mM trehalose) was 100 foldconcentrated by centrifugation and re-suspension in concentratedcryoprotectant mix (as described in WO 2010/124855). CFU count per mlwas determined for the bacterial suspension. The suspension waslyophilized in bulk and in vials (2.5 ml fill volume). For viabilityassessment, lyophilized 2.5 ml vials were reconstituted with 2.5 mlpurified water and CFU count per ml was determined. Viability % wasdetermined from the ratio of CFU before and after lyophilization. 2independent production batches (sAGX0167 and sAGX0309) yield >100%survival after lyophilization.

Both (A) and (B) lyophilized powders were further formulated withsuitable excipients to standardize CFU/g. sAGX0037, sAGX0167 andsAGX0309 were filled in HPMC capsules to a minimum of 1.2×10¹¹CFU/capsule. Capsules were banded with a cellulose film and coated withmethacrylic acid-ethylacrylate co-polymers as an enteric coating film,for targeted delivery to the small intestine and colon.

Trehalose was determined essentially as described in Example 1.

Results

As indicated in Table 3, strains that can accumulate trehalose showgreatly enhanced resistance to drying stress as experienced duringfreeze-drying.

TABLE 3 CFU/ml CFU/ml formulated freeze-dried Trehalose Strain biomasscake content survival sAGX0037 exp. 1 A 1.60 × 10¹¹ 1.26 × 10¹¹ n/a 79%sAGX0037 exp. 2 1.50 × 10¹¹ 1.47 × 10¹¹ n/a 98% sAGX0037 exp. 3 1.40 ×10¹¹ 1.02 × 10¹¹ n/a 73% sAGX0037 exp. 4 1.50 × 10¹¹ 1.26 × 10¹¹ n/a 84%sAGX0085 exp. 1 2.00 × 10¹¹ 1.40 × 10¹¹ n/a 70% sAGX0085 exp. 2 1.60 ×10¹¹ 1.42 × 10¹¹ n/a 89% sAGX0167 exp. 1 B 1.14 × 10¹¹ 1.21 × 10¹¹ 16.29106% mg/g ww sAGX0309 exp. 1 1.21 × 10¹¹ 1.45 × 10¹¹ 16.72 120% mg/g ww

Example 9: Survival During Intestinal Passage Through Porcine Intestine

Experimental

Sows (>150 kg) were surgically equipped with cannulae at the proximalduodenum and proximal colon. In the duodenal cannula, encapsulated,freeze-dried sAGX0037 and sAGX0167 were inserted. Colonic content wassampled from the colon cannula at 0, 2, 4, 6, 8 and 10 hours postadministration. Viability % was determined as the ratio between live(CFU count) and total (live and dead; Q-PCR analysis) L. lactis in thesamples. Numbers are given in Table 4.

Results

Strains that can accumulate trehalose show greatly enhanced survival,independent of the feeding or fasting status, in a large intestinalsystem (pig).

Table 4 and FIG. 8 indicate that when compared to freeze dried andencapsulated sAGX0037 (Table 4 A), freeze dried and encapsulatedsAGX0167 (pregrown for intracellular trehalose accumulation; Table 4 B)show enhance survival during intestinal passage through porcineintestine, both when pigs were fasted for 24 hours (FIG. 8 A) as well asduring ad libitum food availability (FIG. 8 B).

TABLE 4 Time-point sAGX0037 sAGX0167 p-value Fasted T0 — — T2 8.5% 45.1%0.033 T4 3.0% 30.9% 0.001 T6 4.5% 24.8% 0.044 T8 1.9% 10.7% 0.176  T100.0% 13.2% Fed T0 — — T2 — — T4 0.0%   89% 0.001 T6 0.0%   66% 0.173 T80.0%   25% 0.203

Example 10: Trehalose can be Accumulated after Production of Biomass

Experimental

Indicated strains were grown overnight in GM17T (16 hrs at 30° C.) andwere collected by centrifugation (15 min at 4000 rpm). Bacterial pelletswere resuspended in fresh GM17T+500 mM trehalose and incubated.Intracellular trehalose content was determined at T=0, 1, 2 and 4 hoursas described in Example 1.

Results

As indicated in FIG. 9 A, trehalose can be accumulated after biomassproduction, when the bacteria are incubated over time. As indicated inFIG. 9 B, this can be achieved only in trePP KO strains (sAGX0090 vsother) and does not require further gene insertion or deletion(sAGX0169). The additional presence of otsB (sAGX0167, sAGX0346,sAGX0354, sAGX0360) stimulates trehalose accumulation.

Example 11: Maltose can Stimulate the Accumulation of IntracellularTrehalose

Experimental

Indicated strains were grown overnight (ON; 16 hrs at 30° C.) in GM17T,+1-500 mM trehalose, GM17T+0.5% maltose (GMM17T)+500 mM trehalose orM17T+0.5% maltose (MM17T)+500 mM trehalose and were collected bycentrifugation (15 min at 4000 rpm). Intracellular trehalose content wasdetermined as described in Example 1.

Results

As indicated in FIG. 10, maltose stimulates the accumulation ofintracellular trehalose in over night grown cultures.

Example 12: Maltose can be Converted to Intracellular Trehalose Duringor after Production of Biomass

Experimental

Indicated strains were grown overnight (ON, 16 hrs at 30° C.) in GM17T,cells were collected by centrifugation (15 min at 4000 rpm), resuspendedin M17T+0.5% maltose (MM17T and incubated for 8 hours (>8 h MM17T).Alternatively, indicated strains were grown ON in MM17T. Intracellulartrehalose content was determined as described in Example 1.

Results

As indicated in FIG. 11, maltose can be converted to intracellulartrehalose during or after production of biomass.

ABBREVIATIONS ADP adenosine-5′diphosphate anti-TNF Antibody recognizingtumor necrosis factor ATP adenosine-5′triphoshate celBcellobiose-specific PTS system IIC component CFU Colony forming unitCOGs Clusters of Orthologous Groups of proteins eno enolase(phosphopyruvate hydratase) gene (Gene ID: 4797432) gapBglyceraldehyde-3-phosphate dehydrogenase gene (Gene ID: 4797877) Gene IDGene identifier GM17 Oxoid M17 + glucose at 0.5% GM17T Oxoid M17 +glucose at 0.5% + thymidine at 0.2 mM GMM17T Oxoid M17 + glucose at0.5% + maltose at 0.5% + thymidine at 0.2 mM hIL-10 Human interleukin-10HPMC Hydroxypropylmethylcellulose hTFF-1 Human trefoil factor-1 KOKnock-out; gene deletion, gene replacement, gene disruption M maltose at0.5% M17 Oxoid M17 M9 M9 salts (Difco) MM17 Oxoid M17 + maltose at 0.5%MM17T Oxoid M17 + maltose at 0.5% + thymidine at 0.2 mM n/a notapplicable NADP⁺ nicotinamide-adenine dinucleotide phosphate NADPHreduced nicotinamide-adenine dinucleotide phosphate ODx Optical densityat x nm wavelength otsA Escherichia coli osmoregulatory trehalosesynthesis A; trehalose-6- phosphate synthase otsB Escherichia coliosmoregulatory trehalose synthesis B; trehalose-6- phosphate phosphataseotsBA Coupled expression unit for otsB and otsA pgmBβ-phosphoglucomutase (Gene ID: 4797271) PhllA Promoter of the HU-likeDNA-binding protein gene (Gene ID: 4797353) ptcC cellobiose-specific PTSsystem IIC component Ptre trehalose operon promoter PTSphosphotransferase system rpmD Intergenic region preceding the 50 Sribosomal protein L30 gene T thymidine at 0.2 mM thyA Thymidylatesynthase gene (Gene ID: 4798358) TNF Tumor necrosis factor trePPtrehalose-6-phosphate phosphorylase (Gene ID: 4797140) trePTC Putativephosphotransferase genes in the L. lactis trehalose operon (llmg_0453and llmg_0454; Gene ID: 4797778 and Gene ID: 4797093 respectively) TXTime point X hours uidA Escherichia coli beta-D-glucuronidase gene usp45unidentified secreted 45-kDa protein gene (Gene ID: 4797218) wt wildtype ww wet cell pellet weight

The invention claimed is:
 1. A method for internally accumulatingtrehalose in a Gram-positive bacterium, comprising: propagating theGram-positive bacterium in a medium comprising a substrate materialcapable of being fermented by the Gram-positive bacterium, wherein theGram-positive bacterium (i) lacks trehalose 6-phosphate phosphorylase(TrePP) activity and cellobiose-specific PTS system IIC component (PtcC)activity; and (ii) is a lactic acid bacterium (LAB) or Bifidobacterium.2. The method according to claim 1, wherein stress resistance ormanufacturing, processing and/or storage characteristics of theGram-positive bacterium are improved, and wherein the stress resistanceor manufacturing, processing and/or storage characteristics are selectedfrom the group consisting of resistance to acid conditions, resistanceto heat, resistance to salt, resistance to bile salts, resistance todrying, spray-drying, freezing or freeze-drying, and osmotic resistance.3. The method according to claim 1, wherein the Gram-positive bacteriumdoes not contain a functional heterologous trehalose 6-phosphatephosphatase.
 4. The method according to claim 1, wherein theGram-positive bacterium lacks ptcCor wherein ptcC has been partially orcompletely deleted, disrupted, or inactivated.
 5. The method accordingto claim 1, wherein the Gram-positive bacterium overexpresses a geneencoding a trehalose transporter.
 6. The method according to claim 1,wherein the Gram-positive bacterium expresses a heterologous geneproduct.
 7. The method according to claim 1, further comprising drying,freezing, spray-drying or freeze-drying the propagated Gram-positivebacterium.
 8. The method according to claim 1, wherein the culturemedium comprises maltose or glucose or a combination of maltose andglucose, as a carbon source.
 9. The method according to claim 1, whereinthe culture medium substantially does not contain externally addedtrehalose.
 10. A method for formulating a medicament, a food additive, aprobiotic composition, or a starter culture, comprising: (a) propagatinga Gram-positive bacterium in a medium comprising a substrate materialcapable of being fermented by the Gram-positive bacterium, wherein theGram-positive bacterium (i) lacks trehalose 6-phosphate phosphorylase(TrePP) activity and cellobiose-specific PTS system IIC component (PtcC)activity; and (ii) is a lactic acid bacterium (LAB) or Bifidobacterium,and (b) formulating the propagated Gram-positive bacterium into themedicament, the food additive, the probiotic composition, or the startculture.
 11. The method according to claim 1, wherein the Gram-positivebacterium lacks trePP or wherein trePP has been partially or completelydeleted, disrupted, or inactivated.
 12. The method according to claim 5,wherein the Gram-positive bacterium constitutively overexpresses thegene encoding the trehalose transporter.
 13. The method according toclaim 5, wherein the trehalose transporter is an endogenous trehalosetransporter.
 14. The method according to claim 6, wherein theheterologous gene product is a prophylactic and/or therapeutic geneproduct or antigen.
 15. The method according to claim 8, wherein maltoseor glucose or a combination of maltose and glucose is the main or solecarbon source in the culture medium.
 16. The method according to claim4, wherein the Gram-positive bacterium is genetically engineered to lackptcC or have ptcC partially or completely deleted, disrupted, orinactivated.
 17. The method according to claim 5, wherein theGram-positive bacterium is genetically engineered to overexpress thegene encoding the trehalose transporter.
 18. The method according toclaim 11, wherein the Gram-positive bacterium is genetically engineeredto lack trePP or have trePP partially or completely deleted, disrupted,or inactivated.